The molecular and cellular mechanisms that trigger and sustain a regenerative response

Lay summary

The simple act of living comes with a constant barrage of potential risks, often arising as injuries, which can be either minor or traumatic. In addition, many diseases also are the result of or are responsible for impaired tissue function. Finally ageing is generally associated with diminished tissue integrity and function. The emerging field of regenerative medicine aims to ameliorate all of these consequences and repercussions of life by identifying novel means of repairing injured, diseased and aged tissues, with the ultimate aim of promoting full restoration of their original functions. In this way, this field hopes to extend the healthy lifespan of humans. Given that risks of injury, disease and ageing are not limited to humans, we can sometimes turn to other organisms and ask how do they deal with injuries? More importantly we can sometimes turn to organisms that are much better at repairing their tissues following injury and ask how and why do they exhibit such remarkable regenerative capacities? And finally, can we hope to learn enough from them so that we may be able to mimic their high regenerative capacity with the aim of significantly improving our ability to repair and regenerate tissue in the future? This is exactly what we aim to do in our research.

We have recently been exploiting the remarkable capacity of amphibian tadpoles to fully regenerate their tails and limbs following amputation. As part of our studies, we recently discovered that frog tadpoles produce compounds while they regenerate that are normally thought of as damaging to cells. These compounds are collectively referred to as reactive oxygen species (ROS), and include such molecules as hydrogen peroxide, which at relatively high concentrations is used to bleach hair and to kill bacteria. Our findings were particularly interesting in that they showed that frog tadpoles not only produced these compounds following tail amputation, but we also showed that they had to produce these compounds in order for them to be able to regenerate their tails.. We went on to show that, if we did not allow the tadpoles to produce these compounds, the cells in their amputated tails were no longer able to proliferate as normal and they were no longer able to signal to each other as normal. These findings form the basis for this proposal. Here we wish to ask the following questions:

1. How does injury trigger the production of ROS following tail amputation in frog tadpoles?
2. What are the mechanisms that control the level of ROS, such that it does not rise too high, or remain too low for regeneration to proceed?
3. Which genes are controlled by ROS production?
4. How does ROS levels affect the way cells signal each other during regeneration?
5. Is the production of ROS also required for the regeneration of other appendages, such as limbs and fins, and if so, are the mechanisms similar amongst different organisms?

Answering these questions will help us gain a deeper insight into how sustained ROS production promotes regeneration, and whether this role is conserved across different animals. Ultimately we hope that our study will pave the way towards the development of novel therapies aimed at promoting tissue repair and regeneration in human patients, where regenerative potential is normally limiting.

The overarching aim of this proposal is to investigate the molecular, cellular and biochemical events that trigger and sustain a regenerative response following injury. We will first elucidate the molecular mechanisms that trigger the production of ROS following injury and then we will elucidate the mechanisms that result in the sustained production of ROS throughout the regeneration process. We have already established several HyperYFP transgenic lines in Xenopus and zebrafish, which will permit easy assessment of ROS levels during regeneration. Using these transgenic lines we will perform gain and loss of function analyses of various candidate genes (using transgenesis and TALEN technologies), known to be involved in the production or removal of ROS during regeneration. From these studies, we will be able to identify which gene products are required for the sustained production of ROS during regeneration. We also wish to identify the downstream transcriptional targets of ROS during regeneration. For this, we will perform microarray and/or RNA seq experiments during regeneration under attenuated ROS levels. We will then assess the function of some of these ROS-dependent genes during tail regeneration. Furthermore, we wish to identify the direct targets of oxidation, which impacts on intracellular signalling and/or transcription. This will be done using a combination of gain and loss of function experiments, together with targeted thiol proteomic approaches. Finally we aim to uncover to what extent are the various roles of ROS during appendage regeneration conserved across different models of regeneration, including tail and limb regeneration in frog tadpoles and fin regeneration in zebrafish. To achieve this aim, we will use many of the same techniques to manipulate ROS levels during limb and fin regeneration in frog tadpoles and fish, respectively, and we will investigate the outcome of such manipulations on the efficiency of regeneration of these appendages.

Impact Summary

1. CLINICAL IMPACT. Reconstructive and plastic surgeons have a long standing interest in promoting better healing and regeneration in patients presenting with various disfiguring conditions. Findings from our work will therefore interest these clinicians as they may pave the way towards better or more effective treatments aimed at improving the efficiency of scarless wound healing and regeneration in human patients.

2. ECONOMIC AND SOCIETAL IMPACT. The ultimate goal of this research is aimed at identifying novel therapies that will improve regenerative capacity in human patients. For example, it will be of particular interest if our work leads to the appreciation that modulating ROS levels may promote regenerative responses in human patients. Thus the potential societal impact is very large. Furthermore, given that such a therapy will require the involvement of the pharmaceutical industry, such an approach will inevitably lead to significant economic impact. However, given that injuries and diseases result in a very significant economic burden on the healthcare system, identifying therapies that promote a longer, healthier lifespan will have immeasurably benefit, both on the economic and societal health of the country.

3. RESEARCH CAREER DEVELOPMENT. This research will provide an excellent path for career development for the named scientist on the grant. As an example of such career progression, it is notable that I have previously mentored nine postdoctoral scientists, five research assistants and thirteen PhD students, all of whom have continued onto successful science or clinically-related careers across the globe. I highlight seven of these individuals below:
a. Stephen Nutt, Division, Head of Molecular Immunology at The Walter and Eliza Hall Institute of Medical Research, Victoria, AUSTRALIA
b. Odile Bronchain, Maitre de conferences (i.e. equivalent to a Senior Lecturer in the UK), University Paris Sud, Orsay, FRANCE
c. Ross Breckenridge, Senior Clinical Lecturer, UCL, UK
d. Jeffrey Huang, Assistant Professor, Georgetown University, Washington, DC, USA
e. Jun-An Chen, Principle Investigator, Institute of Molecular Biology, Academia Sinica, TAIWAN
f. Jeremy M Sivak, Assistant Professor, University of Toronto, CANADA
g. Karel Dorey, RCUK Fellow and Lecturer, University of Manchester, UK