Dissecting age-related changes to tissue regeneration in a zebrafish model by live cell imaging and spatial transcriptomics

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This project aims to identify age-associated changes to gene expression that alter cell behaviour during tissue regeneration and may underlie muscle weakness in ageing. This will be achieved by combining live cell imaging and genetic manipulations with spatial transcriptomics in a zebrafish genetic model of ageing.Ageing involves a variety of molecular and cell changes that result in impaired function and diminished regenerative capacity. Muscle weakness in ageing results in frailty and diminished metabolic capacity, making it an important therapeutic area for improving health in the ageing population (1). Although many genes showing age-related changes have been identified in muscle and resident muscle stem cells (muSCs) it is not known how these affect cell behaviour during tissue regeneration. The immune system is an important regulator of tissue repair and is dysregulated during ageing (2). How age-associated changes to the immune system intersect with those affecting muscle cells to alter regenerative capacity is not known. It is therefore critical to identify age-associated changes to genes driving cell behaviour during regeneration in order to identify potential therapeutics that promote effective tissue repair without impairing tissue homeostasis. However, tissue regeneration is highly localized to the injury site and its detailed molecular and cellular mechanisms have traditionally been difficult to probe.This project therefore aims to use a host of cutting edge spatial single-cell imaging and sequencing techniques to identify genes regulating cell behaviour during regeneration affected by ageing and determine whether they can be manipulated to enhance muSC function in vivo.Aims of the project are to: 1. define changes to cell behaviour and metabolic activity by immune, connective and resident stem in muscle of telomerase mutant zebrafish larvae during homeostasis and regeneration.2. define how gene expression in defined cell types within regenerating muscle shows spatially altered localisation in telomerase mutants 3. evaluate the functional importance of genes which show altered spatial distribution in regenerating muscle of telomerase mutants for regulating immune and muSC cell behaviourZebrafish will be used as a model organism for identifying changes to cell behaviour and gene expression in regenerating muscle. Telomerase mutants (tert1) show premature ageing phenotypes similar to humans (3, 4). Importantly, we have demonstrated that the muSC response to injury is impaired in tert1 mutant larvae, providing a powerful genetic tool for visualising cell behaviour in ageing. Animals expressing fluorescent proteins in immune and muSCs will be visualised by microscopy at larval stages during regeneration following focal injury performed by a needle. Regeneration is complete within 4 days following such injuries providing the opportunity for combining live cell imaging with molecular profiling of tissues (5). Cell responses to injury will be analysed using Imaris and a number of parameters tested (directionality, instantaneous speed, mean squared displacement) to define an ageing cell Project Approval Form - Nov 205behaviour profile during regeneration. To profile immune cells and muSCs in tissue a selection of ~500 genes will be selected for spatial transcriptomic analysis using multiplexed FISH (6). Genes will be selected from existing RNA Seq datasets generated by the Knight lab from telomerase mutants and ageing animals that are 1) likely to be important for immune cell or muSC behaviour, 2) show changes in tert1 mutants. Spatial expression of candidate genes in regenerating muscle tissue will be obtained by spatial transcriptomics analyses. Selected genes showing tert1-dependent differences in muSCs and immune cells will be functionally evaluated during regeneration using CRISPR/Cas9 mutagenesis (7) or by pharmacological manipulations