Nikolaou Group Research overview of Developmental Neurobiology Lab The overarching aim of research in the DevNeuro lab is to uncover the crucial molecular and cellular mechanisms that drive the formation, growth, and maturation of the nervous system. We delve into how cells differentiate, migrate, and establish intricate functional networks. Our work examines the influence of genetic and environmental factors on brain development, the impact of abnormalities leading to neurodevelopmental disorders, and the consequences of disrupted connections that can lead to neurodegeneration. We primarily use zebrafish (Danio rerio) as an in vivo model organism due to their high genetic similarity to humans (70% shared genes), optical transparency of embryos/larvae for real-time imaging, and rapid development. They are cost-effective, easily housed, and ideal for high-throughput pharmacological screenings, providing a vertebrate model that bridges the gap between cell studies and mammals. Our lab has made great strides in leveraging advanced techniques to position zebrafish as a key model organism for investigating neuronal connectivity and the effects of synaptic mis-wiring or loss. Given the significant anatomical and physiological similarities between zebrafish and humans, the mechanistic insights we uncover in zebrafish will be crucial for enhancing our understanding of human neurodevelopment and associated diseases. As part of this effort, we have developed methods to: Establish genetically modified animals using CRISPR/Cas9 and transgenesis techniques. Investigate the functional response characteristics of neurons in the brain. Analyse neuronal structure and brain architecture through techniques like single neuron labelling, immunostaining, and lipophilic tracing of axons. Locate synaptic proteins and identify synapses. Conduct transcriptomic profiling of single cells and tissues. Assess behaviour and study neural circuit function. Graft/transplant cells or tissues including xenotransplantation to investigate cellular mechanisms of action. Study molecular interactions within cells and tissues. We use quantitative approaches to measure morphological changes in neurons, determine synapse numbers, evaluate gene expression changes from transcriptomic datasets and quantify molecular interactions. Additionally, we employ mathematical modelling to understand the dynamic changes occurring at the molecular and cellular levels in both developing and mature neurons, such as axon growth, neuronal activity, functional connectivity, and RNA transport, among others. A zebrafish transgenic reporter line, showing fluorescently labelled visual neurons including nerve cell projections connecting the retina with the brain (green-coloured nerve fibre tracts) and their target cells in the brain (blue/cyan-coloured dots). Regulation of axonal mRNA processing in health and disease Our current research is heavily focused on understanding the essential role of local mRNA metabolism in axons. This process involves the active transport of specific mRNAs from the cell body, followed by localised translation that supports various functions such as axon growth, branching, synaptogenesis, the assembly of neuronal circuits, and responses to injury and regeneration. Central to these processes are RNA-binding proteins (RBPs), which play a critical role in regulating the trafficking, stability, and translation of mRNAs in reaction to local extracellular signals. While we have a solid understanding of protein-protein and RNA-protein interactions for many RBPs in the nucleus, the corresponding interactions in neurites are still not well understood. Our team has recently made some exciting discoveries regarding the localisation and co-association of RBPs, including several core spliceosome proteins, within ribonucleoprotein complexes in axons. We’ve demonstrated their significance in forming neuronal connections, such as neuromuscular junctions. To delve deeper into this intricate network, we will employ biochemical assays, utilise molecular genetic techniques in zebrafish to establish new knock-in and transgenic lines, and apply imaging approaches. This will allow us to characterise the complex interactions between RBPs and their axonal mRNA targets in vivo, as well as the developmental processes they govern. Mosaic labelling of live neurons within the developing zebrafish brain with GFP. The soma and growing axon are clearly observed. mRNA molecules are labelled in magenta. Numerous RNA-binding proteins (RBPs), especially those involved in RNA splicing regulation, have been observed to mis-localise and form aggregates in the cytoplasm. This mis-localisation can disrupt neuronal function, ultimately resulting in synapse loss and degeneration. Our research, alongside that of others, has demonstrated that various splicing regulators exhibit a bimodal distribution, localising both to the nucleus and neurites. Alarmingly, many of these RBPs have been associated with neurodegenerative diseases such as Alzheimer’s disease and Amyotrophic Lateral Sclerosis (ALS). Another focus of our research group is to investigate how variants linked to these diseases and their mis-localisations affect the normal functioning of these proteins and contribute to the loss of synaptic connections. The structure of an individual motor neuron, characterised through mosaic labelling using fluorescent proteins. Elucidating disease mechanisms and developing novel therapeutics Numerous genes associated with neurological conditions, including neurodevelopmental disorders and neurodegeneration, play a crucial role in neuronal development. To delve into human disease, we are focusing our research on understanding the physiological functions of these disease-linked genes. Our subsequent studies will center on known human variants that pose risks. Our team specializes in creating transgenic animal models and utilizing CRISPR/Cas9 for both knockout and knock-in lines. We conduct comprehensive phenotypic analyses, evaluating mRNA and protein levels, along with the distribution of proteins within tissues—both at cellular and subcellular levels. We also investigate changes in neuronal structure and synaptogenesis. To capture neuronal activity throughout the larval brain, we employ genetically encoded calcium reporters and light-sheet microscopy, allowing us to generate network activity maps for comparison with control neural networks. Additionally, we examine animal behavior to assess the functional outputs of the nervous system. Zebrafish has become a widely used model organism, particularly valued for its cost-effectiveness in high-throughput drug screening and the ease of rapid genetic manipulation using techniques like CRISPR/Cas9. Currently, our team is dedicated to creating disease models that reflect human neurological conditions, such as epilepsy and ALS. We are keen to collaborate with academic institutions and industry partners who are interested in leveraging these genetic models to further understand disease mechanisms and develop effective treatments. If you’re interested in exploring potential collaborations, we’d love to hear from you! Axonal tracts labelled with an antibody against acetylated tubulin. Commissural nerve fibres can be seen crossing the brain at multiple locations (e.g., optic tectum and cerebellum). Current projects Current research projects address the following questions: What roles do RNA splicing proteins play in mRNA processing within axons? In what ways do the extra-nuclear pools of RNA splicing proteins interact with and regulate the axonal transcriptome? How can the mis-localisation of splicing factors and RNA-binding proteins lead to the breakdown of synaptic connections, ultimately contributing to neurodegeneration? What are the molecular and developmental roles of rare epilepsy-linked genes during neurodevelopment? Can our zebrafish disease models be utilised to uncover new therapeutic targets for neurological diseases in humans? Willing to supervise doctoral students We are always open to students interested in neuronal cell biology and neural connectivity mechanisms in both health and disease conditions. PhD funded projects in the lab will appear below when become available. Research staff positions Highly motivated researchers interested in joining our group are always welcomed. If interested, please contact us well in advance for an informal discussion. Current positions will be advertised below when available. Fellowship Applications We are happy to support applications and host recipients of personal research fellowships (e.g., Royal Society University Research Fellowship, BBSRC fellowship, Marie Curie, EMBO). Teaching interests Nikolas is a Senior Lecturer in the Department of Clinical and Biomedical Sciences (Exeter Medical School). His teaching roles span across both undergraduate (UG) and postgraduate (PG) degrees. He delivers lectures on the UG Neuroscience course programme. He supervises final year UG and offers MSc lab projects. He supervises on average 2 PhD students working in his lab. NEU3001 – UG research projects NEU3008 – Frontiers in Neuroscience NEU3009 – Neurodevelopment NEUM006 – MSc Research dissertation projects Past group members Joshua Lloyd-Jones (PhD student 2021-2025) Tilly Baldacchino (Post-doc 2023-2024) Chloe Edwards (RA 2024-2025) – Currently doing a PhD at the The Medical University of Graz