‘I am a Sheffield-based PhD student, funded by the BBSRC White Rose DTP. Previously, I completed a BSc in Biomedical Science, and an MSc(Res) in Translational Oncology at The University of Sheffield. My key research interests lie within improving prognosis and treatment of age-related disease. My PhD involves developing a new way to study an age-associated problem, the accumulation of unhealthy (senescent) cells. We hope that the project will allow us to identify more effective ways of removing these unhealthy cells, to allow our healthy cells to replenish aged tissue and reduce the onset of age-associated diseases such as Cancer, Alzheimer’s Disease, and Osteoarthritis.’
Title: Establishing a novel in vivo model of stress-induced senescence using zebrafish larvae
The main aim of this project was to characterise stress-induced senescence in zebrafish larvae and develop ways of imaging senescence in vivo. The first objective to tackle this involved developing an assay for irradiating zebrafish, which resulted in stress-induced senescence in an early-stage zebrafish. To be able to detect whether senescence was being induced in irradiated zebrafish larvae, numerous techniques had to be developed. This included a combination of quantitative PCR, in situ hybridisation, immunofluorescence, and immunohistochemical approaches initially.
Once there was evidence to suggest that irradiation of zebrafish larvae could result in stress-induced senescence, the next objective was to take advantage of zebrafish larvae as a model organism and develop a transgenic reporter for the well-characterised marker of senescence, p21. This would allow us to identify senescence in a live, whole organism context, at the cellular level, something that hasn’t been possible previously. To make this transgenic reporter, we used BAC transgenesis to develop a zebrafish that would express GFP whenever & wherever p21 was being expressed. Next, we characterised whether the fluorescence from the transgenic reporter zebrafish faithfully recapitulated endogenous p21 mRNA expression. We used our established irradiation assay, fluorescence imaging, and flow cytometry to confirm this. Once characterised, the TgBAC(p21:GFP) zebrafish allowed us to further tackle our objective of providing further evidence that we were inducing senescence. Fluorescence activate cell sorting and immunofluorescence allowed for analysis of several senescence markers within the same cell, and qPCR of the sorted populations identified the presence of the SASP.
We have developed a novel tool that will allow us to help us understand more about how senescence functions in vivo. This study is the first to assess the effects of irradiation on zebrafish larvae in terms of whether it can induce senescence, as is known in mammals. It further adds to the notion that senescence is an evolutionarily conserved mechanism in vertebrates. The development of this transgenic system has allowed for the first work studying the relationship between immunity and senescence at the cellular level, in a whole organism context.
Zebrafish can identify off-target drug toxicities, as well as show whole organism improvements in health in the presence of an efficacious compound. Compounds that remove senescence have been highlighted as a possible approach to extend healthy life span. Currently, there is a bottleneck whereby lots of compounds can be identified through in vitro and bioinformatics approaches, but it is laborious both in terms of time and finances to test all of these drugs in mice. Our zebrafish system can identify off-target toxicities and changes in organism health following drug treatment in a more high-throughput manner.
This system whereby we have been able to induce in vivo senescence in a short period is particularly useful within the ageing field in terms of replacing, reducing, and refining experiments on animals. It should replace alternative animal models in senescence research as it is less costly than maintaining large stocks of ageing animals. It will refine procedures by providing an approach to understand aspects of ageing without lifelong animal experimentation. This zebrafish system should better identify the in vivo efficacy and off target toxicities of novel senolytic drugs also. Compounds identified in this way should be more likely to succeed in pre-clinical trials and reduce the number of protected animals used.