Adult stem cells are undifferentiated tissue specific cells that are able to regenerate damaged tissues. A population of stem cells reside in skeletal muscle and can rapidly repair the tissue upon injury but if muscle stem cell function is perturbed this can lead to fibrosis and muscle weakness. Migration of muscle stem cells to sites of muscle damage is thought to be regulated by signals from activated immune cells, such as macrophages. Muscle regeneration can be evaluated in mouse models by creating an injury to the muscle, typically via injection of myotoxic compounds, cryoinjury or weight bearing exercise. One of the issues with using a mammalian model for these studies is the inability to observe regeneration in real time, as tissue must be examined post-mortem.
Why we funded it
This PhD Studentship aims to optimise a larval zebrafish model of muscle regeneration to replace the use of mice and protected zebrafish1 in these studies.
Muscle regeneration studies currently require multiple animals to be sacrificed at specific endpoints to observe various stages during muscle regeneration. In total, approximately 24 animals would be required to test one experimental variable at four different stages during regeneration. A total of 474 papers were published in 2015 relating to regeneration studies in mouse models, needing approximately 47,000 mice for these studies. Dr Knight and colleagues at King’s College London estimate that the use of zebrafish larvae could replace approximately a fifth of muscle regeneration experiments in mice and protected zebrafish leading to a global replacement of around 10,000 mice per year.
In the proposed larval zebrafish model, an injury to the tailfin muscle will be created using a laser resulting in highly reproducible injuries to minimise the number of fish required in each study. Muscle regeneration will be analysed by confocal microscopy using genetically modified fish so that fluorescently labelled migrating inflammatory cells and muscle stem cells can be tracked in the tailfin. The optical clarity of zebrafish larvae removes the need for euthanasia at specific endpoints instead allowing muscle regeneration to be examined in real time. The newly optimised model will be used to determine the importance of immune cells and migrating stem cells in muscle regeneration and to identify the signals that regulate these processes.
1 The Animals (Scientific Procedures) Act 1986 defines protected larval forms of vertebrate animals as independently feeding. Zebrafish larvae begin independently feeding 120 hours post fertilisation so are not considered protected until this time.
This project will optimise a zebrafish model of muscle regeneration in order to produce high quality datasets detailing cell responses in vivo. This will be achieved by developing a highly reproducible method of injury in the tail of embryonic zebrafish using a laser and observing and measuring the regenerative process by confocal time-lapsed microscopy.
The project aims to understand how genes and signals can affect muscle regeneration by changing the behaviour of muscle stem cell and immune cell responses to injury. Results from this work will therefore reveal how specific factors control cells to enhance or inhibit regenerative.
Currently used cell and animal models for understanding regeneration are limited in a variety of ways: they either do not replicate the complicated situation in a human or do not permit examination of cells as they repair damage. Larval forms of zebrafish are ideal for investigating regeneration, as they possess many of the key cell types found in mammals, they are
transparent, permitting observation of cell behaviour in a living animal and they are able to rapidly regenerate.
A protocol for muscle regeneration in transgenic embryonic zebrafish will be developed using a laser to cause a small injury to muscle. Fluorescently labelled muscle stem cells and macrophage responses to injury will be captured by time-lapsed confocal microscopy and cell responses measured. The importance of pro- and anti-regenerative signals will be tested using genetic and pharmacological means. Candidate genes that may control cell responses to injury will be identified using cell-specific transcriptional profiling, then functionally tested during regeneration.
Principles developed in this project would be widely applicable for investigating regeneration of other cell types other than muscle. Ultimately, results from this project will identify potential candidate molecules to focus on for promoting muscle strength and health in humans.