How are regenerative cells recruited during zebrafish larval tail regeneration?

Regenerative biology is a scientific field that aims to understand the mechanisms and limitations of regenerative capacity in different organisms. Although mammals are able to heal wounds and regrow many tissues such as skin and muscle, the regeneration of complex structures is limited to the liver, kidney and digit tip. Aquatic vertebrates on the other hand, are able to regenerate large portions of organs including limb, tail, spinal cord, retina and heart. When comparable damage occurs in mammals, tissue fails to regrow and scarring occurs.
Organ regeneration in aquatic vertebrates requires many of the same genes that are deployed during the initial development of the organ. This indicates that the reinitiation of developmental genes is important to rebuild the organ. One possible explanation for the poor regenerative potential observed in mammals is that damage does not trigger these developmental genes. These genes are present in mammals but are simply not reactivated. Thus, the elucidation of the mechanism by which tissue damage triggers developmental genes is crucial to further our understanding how successful regeneration takes place.
A second reason for poor mammalian regeneration may be a failure to recruit new cells to the site of injury in sufficient numbers to restore the missing tissue. When tissue is damaged in mammals, limited numbers of regenerative cells are attracted to the injury. Studies in aquatic species indicate that large numbers of regenerative cells can be recruited from a diverse array of sources. The identification of the mechanisms that recruit cells to contribute to regeneration in aquatic species may help to facilitate regeneration in mammals by allowing more regenerative cells to be attracted to the damaged site.
Our project focuses of zebrafish as a model for regeneration. When a small portion from the end of the tail is removed, the fish regenerate the missing tissue after 3 to 4 days. By studying this process, we have identified a crucial regenerative pathway (called the Hedgehog pathway) that recruits regenerative cells to the site of injury enabling the redevelopment of the tail. In the first part of this project we aim to determine precisely which cells are recruited to repair the damaged tail and what roles do they play during regeneration. In the second part of the project we aim to determine how Hedgehog signalling acts on these cells to initiate regeneration.
The knowledge gained from our study will hopefully one day help us to develop new clinical approaches to organ regeneration in humans. Many of the same genes that are active during zebrafish regeneration are found in humans, including those that act in the Hedgehog pathway. This suggests that we may be able to activate the same pathways in humans to mobilise untapped sources of regenerative cells and improve our regenerative capabilities.

Aquatic vertebrates (e.g. salamanders, fish and tadpoles) possess a remarkable ability to regenerate many organs after extensive damage. It is hoped that by studying these species we may learn the mechanisms that enable regeneration. This project builds upon our previous study of zebrafish which found that the Hedgehog pathway links wound healing to redevelopment during larval tail regeneration. However the precise mechanism by which Hedgehog signalling acts is unknown. We now propose to focus on the earliest stages of regeneration to unravel why Hedgehog is required for regenerative cell recruitment and the establishment of the blastema and wound epithelium. The project utilises several powerful transgenic technologies to image and lineage trace regenerative cells in vivo. These include Cre/Lox recombination, Kaede fluorescent protein conversion and time-lapse analysis of cell movements using ubiquitously expressed nuclear fluorescent protein. In addition we will study Hedgehog signalling during regeneration by conditionally activating and inactivating the pathway to determine how regenerative cell behaviour changes and how expression of target genes is altered. Upon completion this project will confer a deeper understanding of the mechanism by which tissue damage triggers regeneration in the zebrafish tail.

In addition to the advances our basic understanding of regeneration, this research will have longer-term impacts within the research community and society as a whole.

Promotion of science to a broader audience -- The astounding regenerative power of aquatic species is a source of fascination for the public. Whole body regeneration observed in planaria is a mainstay of school biology labs around the world. Although the study of regeneration necessitates damage to the organism, the low neural complexity of fish and the rapid and highly efficient repair of larval tail elicits fewer moral objections. Indeed, the majority of our experiments take place before the fish has reached 5.2 days old, the age at which zebrafish become protected by Home Office legislation. Zebrafish research in general captures the imagination of the average person because they are familiar as pets and they are relatively transparent and easy to handle. At outreach events our stalls and "The Amazing Green Fish Pod" are mobbed, and being able to see live fish and their research potential in person is an inspiration for would-be-scientists of all ages.

Improvement to health -- A central goal of regenerative biology is to provide a basic science knowledgebase that will underpin improved clinical approaches to regenerative medicine. Our laboratory aims to understand the signal(s) that recruit cells to contribute to regeneration. If this knowledge is translated into medical approaches then it may facilitate healing in patients that have extensive tissue loss, or in the elderly when tissue repair occurs more slowly. By increasing the number of regenerative cells available during repair we may enhance our regenerative capabilities. A second aspect of our research involves understanding how redevelopment of an organ is triggered. This involves activation of signals involved with patterning, proliferation and morphogenesis. Regrowth of tissues in complex organs such as limbs and heart in human patients is very likely to involve reactivation of these signals just as it does in highly regenerative species. Patient recovery from accidents, amputation, surgery and age-related fragility may all be improved by the application of regenerative and/or developmental signals. Even a modest enhancement in these patients may have great effects on their quality of life as well as reducing care costs over time.

Development of new biotechnology-based approaches to regenerative medicine -- An understanding of the signalling pathways that are involved with organ regeneration may inspire new revolutionary approaches to regenerative medicine. During regeneration in aquatic species the timing and spatial distribution of ligands is tightly regulated. If such signals are to be used in regenerative medicine their delivery will require a similar profile of activation to ensure suitable levels of tissue exposure. Timing the levels of release as well as combinatorial signalling will be required to optimise the benefits of regenerative signalling. Development of such technology will likely involve advanced biomaterial manufacture, an industry in which the UK is a world leader. Ideally such materials would enable clinicians to design release profiles for several different ligands over a period of days or weeks. Coupling this approach with stem cell therapy and synthetic tissue scaffolds promises to revolutionise our ability to treat a broad range of acute and chronic clinical conditions.