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Extracellular vesicles (EVs), once believed to be mere debris shed from cells, are small membranous bags filled with a characteristic repertoire of biomolecules that may redefine our understanding of how life operates through crosstalk in the brain beyond synapses. But have we witnessed how EVs are secreted, received and processed to deliver information from one cell to another in a living organism?

This project seeks to deliver a definitive answer to the as-yet unverified hypothesis through an unprecedented integration of genetic manipulation, bioimaging, and spatial transcriptomics. Previously, zebrafish models visualized in real-time a flow of EVs travelling from one organ to another using a fluorescent protein to genetically label EVs. It is, however, still questionable whether the EV's biomolecular cargo escapes the endosome after uptake and is thus delivered intact to actually trigger molecular responses. 

Using the zebrafish as a model organism, proposed here is not an ordinary approach of EV labelling but cargo-driven genetic recombination of EV-recipient cells as an irreversible marker carved into the genome.Centred around a new method for loading of self-detachable Cre recombinase into EVs, two major challenges are tackled; brain-wide fluorescent mapping of the EV donor-recipient network, and single-cell transcriptomic atlas to spatially resolve and profile individual cells among the same cell types based on the footprint of EV cargo delivery. 

Both approaches are realized through transient/stable transgenesis of zebrafish for which I have appropriate infrastructure and expertise. How exactly EVs drive communication in brain health and disease is scarcely known as yet, awaiting the advent of relevant in vivo models. The success indicator of this project is thus compelling evidence of EV transfer within the brain, establishing a zebrafish model that can in future be used to address clinically relevant questions in neuroscience.