Semaphorin/plexin interactions in regenerative neurogenesis in zebrafish

After spinal cord injury, nerve cells are lost and never replaced. Attempts to replace the lost nerve cells are met with limited success in mammalian models. Alternatively, dormant stem cells, which do exist in mammals, could be reactivated to produce new nerve cells, but these cells do not receive the right signal in the injured spinal cord. In contrast, zebrafish stem cells are reactivated after injury and produce nerve cells that are integrated in the injured spinal cord, and fish fully recover from paralysis. As any injury, spinal cord injury elicits an inflammatory reaction, and we have previously shown that this is necessary for regeneration of nerve cells in the zebrafish. However, the identity of the molecular signals that are received by the stem cells in the injured nervous system are unclear. We have analysed the genes that are expressed in single immune cells and stem cells and found specific matching signal (semaporins) and receptor genes (plexins) to be present. Here, we want to test whether this signal/receptor interaction is crucial for regeneration of nerve cells in zebrafish, by mutating the genes in turn and determine whether regeneration is impaired. This research can thus identify the 'language' in which immune cells communicate with spinal stem cells to bring about regeneration. Ultimately, similar signals may be used in therapeutic approaches to instruct the stem cells in mammals to produce nerve cells after an injury and thus bring about repair.

Zebrafish, in contrast to mammals, show regeneration of neurons after spinal cord injury. The immune reaction to injury promotes regenerative neurogenesis in zebrafish, but how cross-talk between immune cells and neural progenitors occurs is poorly understood. We hypothesize that immune cells signal directly to neural progenitor cells via semaphorins to promote neurogenesis. In pilot studies using single cell RNAseq we determined that reactive macrophages show a 6-fold increase in sema4ab expression and that spinal progenitor cells express the receptors plexinb1a and plexinb1b after injury. We will determine spatiotemporal expression patterns of sema4ab and its ligands, and determine their functional roles by assessing progenitor cell proliferation and neuronal differentiation in mutants for ligand and receptors. To establish whether direct signalling occurs, we will expose neural progenitors to recombinant Sema4ab ex vivo and quantify regulation of neurogenesis-associated genes. To distinguish between effects in immune cell and neural progenitors, we will conditionally ablate plexinb1 function only in neural progenitor cells in vivo and determine regenerative outcome. This project will elucidate at the fundamental level if and how immune cells directly influence spinal progenitor cells after injury. This will provide targets for future interventions in non-regenerating mammals.

The present project aims to elucidate how macrophage-derived Semaphorin 4a directly promotes regenerative neurogenesis via Plexin-B1 receptors on spinal progenitor cells. The data and tools generated will benefit researchers in Neuroscience, Immunology, and Regenerative Biomedicine. Within Edinburgh, our work will strength collaboration with Immune System/Fibrosis specialists, e.g. Drs Neil Henderson (Macrophages in Liver Repair and Fibrosis), and Dirk Sieger. We also plan to exchange data with Prof Liz Bradbury, KCL, who is investigating similar processes in mammalian spinal cord injury. Dissemination/translation is further facilitated through the European NEURONICHE spinal cord repair network that CB coordinates and which will facilitate any translation to mammalian models.

The current proposal may reveal targets for regenerative therapies. We have regular meetings with Dr Emma Mickley, Director of Business Development at Edinburgh University, to discuss commercial potential of our research. This ensures early identification of commercial potential and support for commercialisation funding, any patent application or translation.

We will present our scientific findings at local, regional, national and international meetings of the zebrafish and neuroscience associations (e.g. International Zebrafish Meeting, International Spinal Research Trust, Society for Neuroscience), as well as the EMBO 2020 Regeneration meeting in Castelldelfels, Spain we are organising in 2020 (funding outcome pending).

Our work will be covered in University press releases, which are regularly leading to media coverage (National and international press, BBC Radio 4, BBC Word Radio, BBC Scotland). Press releases are planned for the start of the grant to inform the public about our research. Further press coverage is planned to accompany publications arising from our work.

The Centre for Discovery Brain Sciences is fully committed to increasing public awareness of neuroscience. We sponsor internal and external lecture series including Edinburgh Neuroscience Day, Brain Awareness Week and the Annual Distinguished Lecture, the latter two open to the public. Findings from this grant, in lay language, will be featured on the Centre's and Deanery's web sites.

We regularly participate in public engagement activities, e.g. Centre tours, to explain our science to patients, carers, potential donors and the general public. For example, our postgraduate students have presented our work in the main mall of the New Royal Infirmary (annually since 2014), in Anne Rowling Clinic public events, TB has presented to a Muscular Dystrophy Patient group (2015), and CGB at the local U3A science group event (2018). We will continue these activities and participate in tours of the Centre explaining our work to the public.

Regeneration research is a particularly attractive topic to engage pupils in science. We will participate in Science and Career Fair activities at local primary and high schools and use work from the current grant to underscore the importance of developmental and regenerative biology and the use of animals in research.

The impact activities will be performed by the PIs and our post-graduate students, who present their data at conferences, engage in the public in science outreach activities, participate in the annual three-minute thesis competition, and receive public engagement training through the University and Beltane Network. PI and postgraduate researchers on this grant will present data at the international Zebrafish Meetings (USA 2021, Asian Pacific 2022, Europe 2023) and the Network meeting of the International Spinal Research Trust (2021 or 2022). We will also engage the lay public and scientists at Edinburgh Science Festival Activities.