Theses - Genetics

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Open Access
Tryptophanase Regulatory Mechanisms in Escherichia coli
Kelly, Ellis
Indole is a heterocyclic signaling molecule, synthesized by a wide array of bacterial species, but most studied in *Escherichia coli*. Indole is formed by the degradation of L-tryptophan through the action of the enzyme tryptophanase (TnaA). The *tnaA* gene is part of the tryptophanase (*tna*) operon, which is regulated by catabolite repression and tryptophan-induced transcription antitermination. Recent studies have unveiled two distinct kinetic modes of indole signalling in *E. coli*: a long-lasting but low-level (persistent) signal, and a transient, high-level (pulse) signal. The underlying mechanisms that regulate the indole pulse phenotype have not been fully characterised. Pulse signalling is thought to be linked to the increased expression of tryptophanase during the transition from exponential to stationary phase. Nonetheless, empirical data from the literature indicates the presence of tryptophanase during early to mid-exponential phase when indole synthesis is low or non-existent. This contradictory evidence indicates the existence of additional regulatory mechanisms controlling tryptophanase activity, and the indole pulse. This work has combined conventional shake-flask culture approaches with single-cell analysis to investigate tryptophanase expression and activity across all *E. coli* growth phases. From these studies a hypothesis has emerged that the indole pulse is triggered, at least in part, by post-translational activation of tryptophanase that results in the surge of indole production. Initial experiments focused on characterising the indole pulse. Significant variability was observed in the timing of the indole pulse in shake-flask culture, its timing varying within a window of about 40 min and its duration ranging from 30 to 45 minutes. The inability experimentally to regulate the timing of the pulse by mutation of the *tna* operon or by plasmid-based tryptophanase expression indicated a post-translational regulatory mechanism might be acting on tryptophanase. Tryptophanase expression measured throughout growth showed tryptophanase protein levels *per* cell are similar in exponential and early stationary phase. This suggests the pulse is triggered by both *de novo* synthesis and activation of pre-existing tryptophanase enzyme. Tryptophanase activity assays on enzyme harvested at different growth phases were consistent with this idea. An indication of the mechanism of post-translational regulation came from the use of single-cell microfluidic studies. Within individual cells, tryptophanase was either concentrated at a polar focus or dispersed throughout the cell. The dispersal of polar foci appears to be critical for the triggering of the indole pulse. As a result of the COVID-pandemic, laboratory access was restricted for a substantial portion of this study, prompting the use of alternative research methods. Consequently, an exploration of tryptophanase distribution and potential evolutionary history was undertaken in *E. coli* and other gut microbial species. The results indicate TnaA is maintained in *E. coli*, and there is evidence that it is a component of the core genome. TnaA extends beyond *E. coli*, being identified in 10% of the bacterial species sequenced in the microbiome. It is present across various phyla of the gut microbiome, including Proteobacteria, Firmicutes, and Bacteroidota.
Open Access
A TRIM28 centric strategy to efficiently uncover the contribution of KZFPs to the evolution of gene regulatory networks
Davis, Juliette
The family of KRAB zinc-finger proteins (KZFPs) is the largest group of DNA-binding factors in tetrapods and is rapidly evolving, with >350 protein-coding members in humans. While the individual roles of most human KZFPs is poorly characterized, it is known that their primary role is to epigenetically silence transposable elements (TEs). KZFPs bind TEs in a sequence specific manner and recruit TRIM28 which interacts with other proteins to add H3K9me3, inducing silencing-associated heterochromatin. Interestingly, it was discovered following a large-scale survey of their binding sites that most human KZFPs target evolutionary conserved transposable elements that have long lost their transposition potential. This suggests additional selection pressures aside from their role in preventing transposition of young mobile elements. To explain the long-term conservation of KZFPs and the remnants of their TE targets, it was hypothesized that KZFPs participate in the domestication process of TEs. As they have varied expression patterns, KZFPs were theorized to serve as have a role in the regulation of domesticated TEs in a cell-context specific way. We have recently been accumulating evidence in favour of this model; for example, ZNF808 has recently been identified as an essential primate-specific regulator of pancreas development via the regulation of MER11 elements which, when derepressed, are able to trigger a liver cell fate program. Here I present a new large-scale strategy to uncover links between KZFPs, TEs and gene regulation. We leverage the shared ability of most KZFPs to recruit TRIM28, which is essential for their role as epigenetic silencers. Targeting TRIM28 allows for the indirect capture of the global activity of KZFPs active in a particular context. First, I have produced genome-wide maps of TRIM28 binding using ChIP-exo in multiple different cell types to understand which elements are differentially silenced between cellular contexts. Second, I demonstrate that endogenously tagged TRIM28 with an inducible protein degradation tag allows us to quantify early transcriptomic events unfolding after its depletion. Together these strategies provide evidence that TRIM28 and KZFPs have cell type specific patterns of binding which contribute to gene regulation by controlling the epigenetic accessibility of cis-regulatory elements and allow us to pinpoint promising individual KZFPs for future studies. This system is amenable to be scaled to multiple cell types and contexts and will allow for a global understanding of the contribution of domesticated TEs regulated by KZFPs to human health and biology.
Open Access
Global dynamics of Bordetella pertussis and implications for control
Lefrancq, Noemie; Lefrancq, Noemie [0000-0001-5991-6169]
*Bordetella pertussis* is the main cause of whooping cough, a disease that still infects millions of individuals annually despite widespread vaccination. This is particularly problematic in neonates, where infection can lead to death. Endemicity, subclinical infections and circulation of multiple lineages hide the underlying dynamics of *Bordetella pertussis* from surveillance systems. Therefore, the extent of its spread across spatial scales remains unknown, as does the role of vaccines in driving changes in strain fitness. In this thesis, I investigate the spatial spread and fitness changes of *Bordetella pertussis* through the development of mathematical models that integrate the information provided by genetic data. I start by investigating *Bordetella pertussis* geographical spread. I found substantial diversity within communities, consistent with widespread subclinical transmission. I quantified the rate of *Bordetella pertussis* spread across spatial scales and found that it spreads between countries and between continents in just a few years. I then investigated the fitness of *Bordetella pertussis* genotypes and the impact of vaccine type switch. I developed an analytical framework that quantifies the relative fitness of different circulating genotypes through time. I found that the implementation of acellular vaccines was associated to large-scale changes in fitness and can explain long-term genotype dynamics. Finally, I explored *Bordetella pertussis* fitness beyond its known genotypes. I developed an agnostic framework that summarises the changes in population composition in phylogenetic trees through time. It allows for the automatic detection of circulating lineages based on differences in fitness, which can be quantified and linked back to specific mutations. I applied this method to *Bordetella pertussis* genomes from four countries and detected the presence of multiple co-circulating lineages with underlying differences in fitness, none of which have been previously identified. The insights gained into *Bordetella pertussis* spatial dynamics and its interactions with vaccine-induced immunity are highly relevant to vaccination policies. This thesis addresses fundamental questions about drivers of spread and fitness that are relevant across disease systems and control efforts.
Embargo
The evolution of ribosomal protein paralogues and their roles in development
Grobicki, Katarina; Grobicki, Katarina [0000-0002-1800-2763]
All cellular protein synthesis - which is indispensable for cell growth, proliferation, and viability - is carried out by ribosomes. The production of sufficient functional ribosomes is essential for development and homeostasis, and reductions in the abundance of functional ribosomes can lead to developmental diseases known as ribosomopathies. Historically, translation has been viewed as a constitutive process, with ribosomes being machines with little to no independent influence on gene expression. However, translation is now known to be highly regulated, and growing evidence has shown that ribosomes can be heterogeneous in composition and may contribute to this regulation. In eukaryotes, cytoplasmic ribosomes consist of two large ribonucleoprotein subunits, 40S and 60S, which are composed of four different ribosomal RNAs (rRNAs) and 79 ribosomal proteins in total. Ribosome heterogeneity can arise from chemical modifications of rRNA, post-translational modifications of ribosomal proteins, incorporation of different ribosomal protein paralogues, or interaction with different ribosome-associated factors. This heterogeneity has been hypothesised to underlie the existence of “specialised ribosomes”, which could exist in specific tissues or developmental stages and may have the potential to preferentially translate specific subsets of mRNA transcripts. However, while many examples of ribosome heterogeneity have been described to date, there are very few examples to date where this has been conclusively linked to functional differences in development. As a result of gene duplications, the *Drosophila melanogaster* genome encodes 14 pairs of ribosomal protein paralogues. Within these pairs, one gene (referred to henceforth as “canonical”) is usually expressed ubiquitously and is essential for viability: mutations in these genes cause homozygous lethality, while heterozygotes usually display the *minute* growth phenotype. At the outset of this project, little was known about the “non-canonical” paralogues, however a number of them have been shown to be expressed in a tissue-specific manner, frequently being enriched in the germline. The aim of this thesis is to systematically examine the duplication of ribosomal protein genes within the *Drosophilidae*, and the potential roles of the 14 “non-canonical” ribosomal protein paralogues in *D. melanogaster*. To characterise the patterns of ribosomal protein gene duplication in *Drosophila*, I designed a pipeline in collaboration with Dr Daniel Gebert to identify duplications of “canonical” ribosomal protein genes in 12 *Drosophila* species, which span 70 million years of evolution. I identified 388 independent duplication events, with most taking place by retroposition. My results indicate that duplicates generally appear to be short-lived, although some have persisted for over 70 million years. To examine the role of each of the 14 “non-canonical” ribosomal protein paralogues in *D. melanogaster*, I used CRISPR/Cas9 to systematically mutate each gene. Phenotypic analysis of the mutants revealed that none of the 14 “non-canonical” paralogues are required for viability or induced the *minute* phenotype. Given their tendency to be expressed in a germline-specific manner, I examined the fertility of the mutants. For most genes, fertility was unaffected in mutants, except for mutations in *RpS5b*, which led to female sterility. I show that loss of *RpS5b* results in strong activation of the Tor pathway and remodelling of germline metabolism. In addition, my experiments reveal that this germline stress response is transduced to the neighbouring somatic epithelium, leading to overgrowth, disorganisation, incomplete Notch activation, and non-autonomous activation of Tor kinase. I show that, in conjunction with the metabolic changes, these phenotypic alterations trigger the activation of the mid-oogenesis checkpoint, resulting in germline death and complete female sterility. Interestingly, the “canonical” *RpS5a* and the “non-canonical” *RpS5b* genes have different and somewhat complementary expression profiles during germline development. Therefore, I set out to test whether the phenotypic alterations in RpS5b mutants result from either the loss of a “specialised” RpS5b-containing ribosome or, rather, from a lack of RpS5 protein in general. To do so, I used CRISPR/Cas9 and homology-directed repair to seamlessly replace the entire coding sequence of *RpS5b* with that of *RpS5a*, and vice-versa. These experiments definitively demonstrated that RpS5a and RpS5b proteins are functionally equivalent during oogenesis, revealing that the *RpS5b* mutant phenotype results from a general lack of RpS5 protein, rather than a potential functional divergence between these paralogous proteins. Altogether, my work highlights how ribosome abundance must be examined whenever investigating potential cases of “specialised” ribosomes.
Embargo
Insights into the piRNA pathway through 16 strains of mice
Meena, Narendra
The non-coding genome generates several classes of non-coding regulatory RNAs. One such class of small RNAs - PIWI-interacting RNA (piRNAs) - are exclusively expressed in the germlines of mammals. This thesis provides an exhaustive bioinformatic exploration of piRNA biogenesis and dynamics utilising a large dual-sequencing dataset, which encompasses bulk RNA sequencing and oxidised small RNA sequencing from whole testes. The study encompasses data from 16 unique inbred strains, 12 classical inbred strains and 4 wild inbred strains, studied across three pivotal developmental timepoints in germ cell development and piRNA biogenesis: E16.5, P12.5, and P20.5, in biological triplicates. Through a differential analysis of bulk RNA sequencing, I delve into the complex pathways of piRNAs, examining the expression patterns of related developmental marker genes related to the piRNA pathway and identify strain-specific differences. Notably, gene expression differences indicate that the three wild inbred strains out of four (WSB_EiJ, CAST_EiJ, and SPRET_EiJ) and two classical inbred strain (BALB_cJ and NOD_ShiLTJ) may have a slower developmental trajectory compared to the classical inbred strains, in agreement with modest differential piRNA length distribution dynamics. In addition, CAST_EiJ, PWK_PhJ, and SPRET_EiJ were observed to have the highest counts of unique strain-specific piRNAs, including Transposable Element (TE)-mapping piRNAs across fetal pre-pachytene piRNAs, postnatal pre-pachytene piRNAs and pachytene piRNAs. Overall, TE-mapping piRNAs predominantly overlap with LINE and LTR transposon elements. Using Trinity RNA transcript assemblies with piRNA mapping, I identify previously undiscovered piRNA precursors and clusters for each strain, with a median count of piRNA precursors for E16.5 being 2206, 3240 for P12.5, and 504 for P20.5 across all 16 strains. This both reaffirms previously known piRNA precursors and broadens our understanding of potential piRNA sources and strain-specific diversity. This research also found that the insertion of the IAPEY4_L LTR/ERVK in the NOCT region is associated with the expression of piRNA precursors, as evidenced by their presence in 7 specific mouse strains (C57BL/6NJ, BALB/cJ, A/J, DBA/2J, AKR/J, NZO/HlLtJ, NOD/ShiLtJ) where these transposable elements (TE) are found. Conversely, strains lacking this TE insertion do not show similar expression patterns. In sum, this work offers new perspectives on piRNA dynamics and their influence on germ cell development across 16 diverse mice inbred strains.
Open Access
Responses to transposon activity and genome damage during germline development
Jansen, Gloria; Jansen, Gloria [0000-0003-3241-6249]
The germline is the cell lineage that is responsible for the inheritance of genetic information in multicellular eukaryotes. In most animal species, germ cells are set aside from somatic lineages during early embryonic development and follow a unique developmental programme that culminates in the production of haploid gametes carrying the genetic material that is passed to the next generation. Due to its central role in genetic inheritance, the germline is known to be the battleground where genetic conflicts between selfish genetic elements, like transposons, and the host genome take place. Excessive transposon activity fulfils the selfish drive of transposons to increase in copy number in the host genome but can impair genome integrity and functionality, thereby threatening the faithful transmission of genetic information. How transposon activity affects the genome and how this impacts germ cell functionality and viability remains poorly understood. This thesis examines responses to transposon activity and genome damage during germline development. I use a classic example of transposon-induced sterility in Drosophila, P-M hybrid dysgenesis, as a model. I show that excessive activity of the P-element transposon in embryonic primordial germ cells (PGCs) leads to the accumulation of DNA double strand breaks (DSBs) and sustained cell cycle arrest prior to fully penetrant germ cell loss during early larval stages. Using a genetic screen for suppressors of the dysgenic sterility phenotype, I identify factors involved in cell cycle regulation and DNA damage responses that play a role in the process leading to germ cell loss in dysgenesis. I then develop a novel approach to characterise new P-element transposition events in PGCs genome-wide and at single-cell resolution. Contrary to the prevailing model that germ cell death is caused by high numbers of new transposition events into coding regions, I demonstrate that dysgenic PGCs acquire few new P-element insertions in gene promoters and introns prior to germ cell loss. I then explore the alternative hypothesis that germ cells are sensitive to the genome damage caused by transposon activity. Using engineered, Cas9-based systems, I show that inducing DNA DSBs at endogenous, silenced P-elements or other, non-coding sequences is sufficient to induce complete germ cell loss during development independent of gene disruption. Indeed, I find that both PGCs and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis despite accumulated genome damage and adverse effects on the development of the next generation. Finally, I investigate tolerance to DSB dosage in somatic cellular domains. Collectively, the findings presented in this thesis demonstrate the existence of developmentally regulated, dosage-dependent DNA damage tolerance thresholds that, on the one hand, safeguard genome integrity during germline development, while on the other hand forming a selective barrier that may shape transposon proliferation strategies. This work serves as a foundation for further study of how responses to genome damage in the germline influence genetic conflicts.
Open Access
Primate-specific KRAB zinc finger proteins and their targets rewire regulatory networks related to inflammation and pancreas development
Triantou, Athina Nikoleta
Transposable elements (TEs) comprise about 50% of the human genome. While a few are still active and can accumulate copies in new locations of the genome, most of these mobile elements have invaded the genome of our ancestors a long time ago in successive waves during evolution. The largest subfamily of DNA binding factors in human, KRAB Zinc Finger proteins (KZFPs), leads to the epigenetic silencing of their bound loci and have been recently shown to mainly target repetitive regions of the genome, mostly TEs. Interestingly most TEs targeted by KZFPs have lost their ability to transpose and pose no threat to the host’s genomic integrity. TEs are known to contain docking sites for host’s transcription factors and contain transcription regulatory elements including promoters, enhancers and silencers. The differential expression patterns observed for members of the KZFP subfamily amongst different biological contexts, have led us to hypothesise that KZFPs participate in gene regulatory networks by toggling chromatin accessibility on their targeted regions. With approximately 400 KZFP protein coding loci in human genome and only a few functionally described, in this thesis we aim to characterise the function of two KZFPs, ZNF808 and ZNF267. ZNF808 was found to be homozygous knock out in patients diagnosed with pancreatic agenesis, presenting neonatal diabetes with insufficient pancreatic exocrine function. Expression and ChIP data for ZNF808 show that it is upregulated during the early stages of pancreatic development and binds on members of primate conserved MER11 elements leading to their epigenetic silencing. On the other hand, ZNF267 is already highly expressed in monocytes and macrophages but it is further upregulated by inflammatory stimuli. ZNF267 ChIP data show that it mainly targets and silences members of the primate specific THE1 and MSTA TE families. Interestingly, despite both KZFPs being primate specific along with their targeted TEs, they are involved in important biological processes. Our study shows that ZNF808 is implicated in the orchestration of gene regulatory networks involved in pancreatic development whereas ZNF267 demonstrates a profile of transcription regulator and promoter/enhancer silencer in monocytes and macrophages. Notably, ZNF808 and ZNF267 expression is regulated by the same TFs which bind on their targeted TEs suggesting a complex gene regulatory network related the respective biological processes. CRISPR knock outs created for ZNF808 in H1 cell line demonstrate changes in their transcriptome where genes implicated in liver development are upregulated during the early stages of their differentiation to pancreatic cells. These changes are coupled with epigenetic activation of nearby MER11 elements known to be bound by ZNF808. Observations in H1 cells were also confirmed in patient derived ZNF808 knock out iPSCs. Similarly, we generated ZNF267 CRISPR knock outs in monocytic cell line U-937 and studied their transcriptome and epigenome as well as in macrophages treated with LPS. Notably, in both cellular contexts we observed transcripts emerging from THE1 elements in the knockouts, suggesting that ZNF267 silences promoter potential of these elements. This thesis presents the contribution of KZFPs in the evolution of novel gene regulatory networks related to key biological processes.
Open Access
DNA methylation variability at the crossroads of stochasticity, genetics, and environment
Kessler, Noah; Kessler, Noah [0000-0002-2740-6663]
During mammalian development, DNA methylation at most CpG sites throughout the genome is erased; these marks are then re-established later. In humans and in mice, typically all copies of a given CpG site in a particular tissue are methylated in all individuals; a large minority are unmethylated in all cases. A substantial proportion are methylated in some but not all tissues. A smaller minority of CpG sites are only partially methylated, and amongst these a proportion have consistent methylation levels across various tissues but variable methylation levels between individuals. Loci exhibiting this latter phenomenon, often referred to as metastable epialleles, were initially identified in inbred mice, where a lack of genetic variability indicates the presence of a genuine epimutation. Furthermore, the consistency of methylation across multiple tissues suggests a timing of methylation establishment which is early in development. These initial findings were located at IAP elements, a murine-specific class of ERV transposable element. There are approximately 10,000 IAP elements in the mouse genome, but the annotation of these elements is often poor and fractured. I developed an ad hoc algorithm to systematically fix the annotations, which reduced the proportion of fractured IAP elements from 36% to 19%. Using a genome-wide dataset, I updated an existing screen of variable methylation at IAP elements and identified that there are several non-IAP ERVs and a LINE element which exhibit metastability. Identifying genuine epiphenomena in humans is challenging due to the lack of individuals having a shared homogeneous genetic background outside of monozygotic twins. Previously, using public data, I performed a screen for variably-methylated loci and found hundreds of loci showing methylation variability in the absence of local genetic variants. Methylation at these loci was then assessed in existing blood samples from over 500 children located in a region of The Gambia where annual seasonal changes in nutritional availability are known to affect circulating levels of metabolites involved in the DNA methylation pathway. Using this experimental design, I identified a testis-specific promoter having methylation which follows a date-of-conception pattern in males, indicating a possible readout of maternal nutritional status during early development. Genotype data from over 1 million SNP loci known to be prevalent in African populations was available for the majority of the individuals described in the above study. I identified that at a variably-methylated promoter of the PAX8-AS1 lncRNA gene, nearby genetic variants could restrict population-level methylation variability: individuals homozygous for the minor allele (haplotype) were all fully methylated while there was a wide range of methylation states among individuals homozygous for the major allele. I termed this phenomenon methylation variance QTL (mvQTL), and then looked for its presence throughout the genome. While canonical methylation QTL (where in a variant causes change in methylation averages but not variance) is common, the mvQTL phenomenon is confined to fewer loci. Through simulations, I found that a small number (4 to 5) of independent genetic variants which collectively determine a CpG's methylation state are sufficient to produce a near-continuous distribution of methylation. Combined with the discovery of mvQTL, these results indicate that existing human DNA methylation association studies may be severely discounting the role of genetics in driving apparent epigenotype-phenotype associations.
Restricted
The role of tryptophanase in biofilms of uropathogenic Escherichia coli
Croft, Cameron; Croft, Cameron [0000-0002-1112-7520]
[Restricted]
Open Access
WNT signalling in the patterning of human gastruloids
Mantziou, Veronika
The study of early human development poses a challenge due to technical and ethical limitations, particularly during the initial four weeks following fertilisation. During this critical period, the fundamental aspects of the body plan are established through a process known as gastrulation, which begins 14 days post fertilisation. Because of the 14-day rule imposed on human embryo research, no experimental studies have been conducted beyond this date. The derivation of human embryonic stem cells (hESCs) has opened up numerous opportunities for studying early cell fate decisions and signals involved in this process. In particular, these cells have enabled the development of embryo models that allow the exploration of various aspects of early development. One of these models is the ‘gastruloid’ which results from the aggregation of small numbers of hESCs and leads to the formation of the body plan. WNT signalling, an evolutionary conserved pathway, is known to play a crucial role during gastrulation with its dysregulation leading to various developmental abnormalities and diseases. This thesis examines the role of WNT signalling in the patterning of human gastruloids. By applying different concentrations of WNT agonists, it is shown that the levels of WNT signalling affect cell differentiation as well as elongation and morphology of the gastruloids. Higher WNT levels induce Brachyury expression, whereas lower favour SOX17 expression, triggering the morphogenesis of SOX17+ tube-like structures. Through these experiments, it is further revealed that human gastruloids might recapitulate the mode of endoderm specification recently observed in mouse embryos. Additionally, this thesis indicates that specifically non-canonical WNT signalling affects the elongation of gastruloids. These responses highlight the high sensitivity of the system to the signalling environment. In addition to WNT signalling, BMP and NODAL also act as critical molecular pathways during gastrulation. This thesis shows that BMP and NODAL can affect cell differentiation and morphology in gastruloids and suggest that human gastruloids require a balance of BMP, WNT and NODAL signalling for their generation. Due to the tractable nature of the gastruloid in vitro model, more factors apart from signalling molecules can be manipulated to study the effects of various stimuli on cell behaviour. One such factor includes the initial cell density. In this thesis, it is suggested that small variations in the standard size of the human gastruloids result in aggregates that exhibit scalable self-organisation, an ancestral feature present in most embryos. By increasing the variation in aggregate size, there was also a concomitant increase in the variation of gene expression. The robust recapitulation of gastrulation-like events in combination with the easy manipulation of the gastruloid system, make it also a powerful tool for teratogenicity assessment. In a first proof-of-concept study presented finally in this thesis, application of a reference panel of seven different compounds results in gastruloid morphological and gene expression changes dependent on the compound and its concentration. Human gastruloids present a powerful system. They can be utilised to explore the mechanisms of human embryo patterning and discern unique features of early development that would otherwise be impossible due to limitations associated with human embryo research. Human gastruloids further have fundamental implications in biomedical research.
Open Access
The spread and control of dengue and chikungunya viruses
Ribeiro dos Santos, Gabriel; Ribeiro dos Santos, Gabriel [0000-0003-4668-8755]
Mosquito-borne viruses such as dengue and chikungunya viruses continue to cause a substantial burden on public health globally. After decades with few tools to tackle the spread of these viruses, new interventions are becoming available. The release of Wolbachia infected mosquitoes has been shown to reduce the incidence of dengue infection. In addition, the first chikungunya vaccines will soon be licensed. However, we currently have a poor understanding of the underlying transmission dynamics of both chikungunya and dengue, and whether these dynamics differ across spatial scales. This means the potential impact of these interventions is unknown. In this thesis, I tackle critical knowledge gaps in our understanding of the transmission dynamics of dengue and chikungunya viruses across different spatial scales. I also provide estimations of the impact of new interventions, providing an avenue to their future widespread use. This thesis brings together high quality data from cohort studies, seroprevalence studies and disease surveillance systems, with sophisticated analytical approaches to answer public health relevant questions. In the first part of my thesis, I work with data from a long running cohort study in Kamphaeng Phet province, Thailand. I used serological data from the study to explore the underlying spatial heterogeneity in dengue virus infection. I found that, in that province, dengue was transmitted consistently from one year to another with a very high rate of infection compared to other places where dengue is found. There additionally was very little spatial variation in terms of force of transmission. I showed that only around 1\% of all infections get detected by disease surveillance systems, highlighting the significant hidden burden of infection from the virus. I also explored potential drivers for transmission in these settings and found that most variation in protection against dengue was at the household level and that door screens had a significant protective effect. In the second part of my thesis, I present the results from a large Wolbachia release project in Rio de Janeiro, Brazil, where millions of Wolbachia infected mosquitoes were released in part of the city. Underlying heterogeneity in where and when cases of dengue and chikungunya were detected had complicated the efforts to understand the impact of the release program. I developed a spatially-explicit analytical approach that characterises the underlying spatial distribution of dengue and chikungunya cases within the city and estimates the impact of the release program on incidence. I showed that despite only intermediate levels of introgression in the city, there was a significant reduction in the incidence of both viruses. In the third section of the thesis, I estimate the global burden of chikungunya virus. I conducted a literature review to identify countries that have local transmission of chikungunya. Then, based on a series of seroprevalence studies across 26 countries, I developed serocatalytic models that estimated the history of infection in epidemic and endemic settings. I show that 113 countries have experienced chikungunya transmission. In a subset of 11 countries, the virus circulates endemically. I estimate that there are 26 million annual infections and 19,000 annual deaths globally, with the greatest burden in the WHO regions of Southeast Asia, Africa and the Americas. The first chikungunya vaccines are currently being licensed. In the last part of the thesis, I develop a simulation-based method to critically assess the impact of different vaccination strategies for chikungunya. In particular, I assess the potential use of a vaccine stockpile in epidemic countries, as is used for other pathogens such as cholera and Ebola. For each country where chikungunya circulates, I present country-specific estimates of infections, cases and deaths averted for different vaccination campaign scenarios. This thesis contributes to the current landscape of infectious diseases epidemiology by exploring the dynamics of transmission for dengue and chikungunya, whose burdens are one of the current biggest public health challenges. It has been written during a critical period of time where vaccines are about to be commercially available and there is an urgent need for better understanding of both pathogens dynamics. Finally, it introduced a wide range of model frameworks that can be applied and built upon to refine our knowledge about arboviral diseases. Much of my work has been conducted in close collaboration with organisations such as the Gavi Alliance, CEPI and the World Mosquito Program. This close relationship with key international bodies involved in the rollout of interventions maximises the impact of my findings.
Open Access
Fitness Landscapes, Genetic Interactions, and the Fitness of Hybrids
Schneemann, Hilde; Schneemann, Hilde [0000-0002-7295-9734]
When two genetically differentiated populations or species come into contact and interbreed, their hybrid offspring will contain a mosaic of the genetic variants characterising the parental lineages, re-arranged into novel combinations. The fitness of these hybrids is central to the evolution of reproductive isolation, but also plays an important role in conservation policy, and in crop and animal breeding. Fitness landscapes are simple mathematical models that generate a rich variety of context-dependent genetic interactions, making them a useful tool for studying the ways in which these interactions affect hybrid fitness. In this thesis, I will explore a particular fitness landscape model based on Fisher’s geometric model (Fisher, 1930), which provides a flexible yet tractable framework for modelling hybridisation. Throughout, I complement the analytical and simulation results with applications to published empirical data. First, I explore the fitness of F1 hybrids, and show how phenotypic dominance can generate a diverse range of outcomes. As the dominance effects at different loci are rarely expressed together during divergence, they are unlikely to be co-adapted. I show that, as a consequence, dominance generally reduces F1 fitness, closely resembling the effects of uniparental inheritance, Still, I predict the effects of dominance can also be beneficial, and this may help to explain transgressive hybrids that prosper in extreme environments. Next, I present results for hybrids of any type, based on a new and more general derivation of the model. I show that predictions can be expressed in terms of two distance measures capturing the net effect and total amount of evolutionary change in terms of additive and dominance effects, as well as their interaction. Each of these terms carries information about the history of divergence, telling us about the type, direction, and subject of selection respectively. Thinking about the long-term outcomes of hybridisation, I then investigate what we can learn from introgression line studies about coadaptation between alleles and the fixability of heterosis. Consolidating the classical theories of heterosis, I illustrate how this model generates complex genetic architectures characterised by transient overdominance. Finally, I present an extension of the model to arbitrary ploidy which lets us investigate the effects of dosage on hybrid fitness. Applying these predictions to published data, I show how they can help to explain repeatedly observed differences in patterns of heterosis and inbreeding depression between diploids and tetraploids.
Open Access
Insights from serological surveillance in infectious disease epidemiology
O'Driscoll, Megan; O'Driscoll, Megan [0000-0002-7972-5703]
Traditional infectious disease surveillance methods rely on testing of symptomatic individuals. However, such methods are often poor indicators of the true burden of infection in a population due to varying rates of subclinical infections, non-specific symptoms, and heterogeneity in health seeking behaviour. Serological surveillance, which tests for immune markers such as antibodies generated in response to an infection, provides invaluable information relating to the underlying burden and dynamics of infectious diseases. In this thesis I developed and applied analytical methods to maximize inferences from IgG antibody data and investigate epidemiological questions relating to the transmission and immune dynamics of human pathogens. Using SARS-CoV-2 seroprevalence studies and age-specific COVID-19 death data, I refined estimates of SARS-CoV-2 infection fatality ratios and inferred infected population proportions in multiple countries. Using longitudinal dengue antibody data from Thailand I reconstructed the dynamics of maternal dengue antibodies and found that antibody-dependent enhancement mechanisms were best able to explain observed age patterns of infant dengue hospitalizations. I developed an analytical framework for the analysis of multi-pathogen serological data to disentangle the antibody responses of related, cross-reacting pathogens. I conducted simulation testing of model performance and applied this framework to serological data for ten arboviruses in Bangladesh. I inferred the levels of between-virus antibody cross-reactivity and reconstructed the spatiotemporal transmission dynamics of each present virus, revealing heterogeneous burdens even at small spatial scales. Overall, serological surveillance represents an important opportunity to fill the gaps left by traditional disease surveillance methods, allowing an increased understanding of pathogen transmission dynamics, rates of pathogen severity, antigenic landscapes, and immune correlates or mechanisms of disease risk and/or protection. The work of this thesis demonstrates how modern analytical methodologies can maximize the epidemiological inferences made from serological data and inform relevant public health questions.
Embargo
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.
Embargo
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.
Open 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.
Restricted
Characterisation of gastruloid-derived Primordial Germ Cell-like cells and investigation of their specification, tissue interactions, and maturation
Cooke, Christopher
[Restricted]
Embargo
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.
Open Access
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.
Open Access
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.

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