The zebrafish tectal stem cell niche - a new model for in vivo analysis of neural stem cell biology

Stem cells in post-embryonic organisms regulate growth, regeneration and repair. The two best-characterized examples in mammals are intestinal stem cells, which renew the gut epithelium every few days, and haematopoietic stem cells that ensure the steady supply of new blood cells. Neural stem cells have acquired prominence due to the therapeutic promise they carry. Theoretically, such cells provide the possibility to regenerate damaged neural tissue thereby providing treatment for neurodegenerative disorders (such as Alzheimer's and Parkinson's disease) and nervous system injuries. In part for this reason, significant efforts are being made to identify and characterise the factors that regulate the maintenance and differentiation of neural stem cells. The mammalian nervous system contains only limited populations of neural stem cells, and these reside in niches that are not easily accessible. It is therefore almost impossible to study such cells in their natural context of the living animal. In contrast to mammals, other vertebrates, including fish, have many populations of neural stem cells making them good models that can complement mammalian and tissue culture experiments. In this project, we propose to establish the zebrafish optic tectum as a new model system to study the biology of neural stem cells. The optic tectum is an important visual centre that grows and adds neurons throughout the life of the fish. The stem cells that make this growth possible, and that have the capacity to generate more than a dozen tectal neuron types, reside close to the superficial surface of the brain. Coupled with the transparency of the zebrafish embryo, these cells can be followed in vivo. Relatively little is known about the behaviour of neural stem cells and progenitors in the intact animal and one of our key goals will be to observe these cells as they divide, move and differentiate. To do this, we will use sophisticated transgenic tools that allow us to visualise cell shape, to follow cell divisions and to track cell movements as progenitor cells generate neurons. Our second goal is to characterise the signals that influence the biology of the neural stem cells. Much has been learned about the signals that control haematopoietic stem cells, and we have found some surprising similarities between tectal and haematopoietic progenitor cells. The Wnt pathway is activated in both locations and we will determine how this pathway influences the maintenance of tectal stem cells, the proliferation of progenitors and the production of neurons. Secondly, recent data has implicated prostaglandins as being novel, critical regulators of the haematopoietic stem niche. We have preliminary data that prostaglandins may have conserved function in the tectal stem niche and will explore this possibility. These studies will help to determine the extent of conservation of the mechanisms that regulate stem cells and will elucidate the signalling mechanisms that underlie neural stem cell biology.A further goal is to resolve how growth is coordinated between the eyes and the optic tectum and we will address this issue by assessing how tectal innervation by the eyes regulates stem cell and progenitor behaviour. The benefits from our work include a better understanding of neural stem cell biology drawn from studies in the intact animal. The transparency of the zebrafish coupled with the ease of accessibility of the tectal stem cells provides a clear advantage for studying neural stem cells in this context. Our research will benefit others working on different stem cell models in helping to design experiments and manipulate stem cells for therapeutic purposes. The signalling pathways that regulate stem cells can be modulated by drugs and/or genetic manipulation and this will provide routes to manipulate stem cells. The proposed research will also establish a new model system to study stem cells that we hope will be exploited by other researchers.

A central goal in stem cell research is to elucidate the mechanisms that regulate maintenance of stem cells, proliferation of progenitors and production of differentiated cells. Existing experimental models have characterised specific aspects of stem cell behaviour and some of the signalling pathways implicated in these processes, but our knowledge of neural stem cells in the intact brain remains sparse or incomplete. This reflects both the scarcity and inaccessibility of stem cell populations in the mammalian brain. In zebrafish, neurons are added throughout life to both the eyes and the optic tectum. The putative stem cells and proliferative cells of the larval tectum are situated directly beneath the epidermis, making them ideally suited for in vivo imaging. Our first approach will be to use novel transgenes to follow the cell-cycle kinetics of the stem/progenitor cells, track their shape changes, polarities, divisions, and movements. These studies will help us to understand the normal behavioural characteristics of stem cells and progenitors in their natural environment. Our second approach will be to determine the effects of Wnt signalling upon the behavioural parameters that we define. We know that this pathway is activated in the neurogenic zone and we will use genetic and pharmacological tools and resources to resolve how this pathway affects the tectal stem cells and progenitors. Prostaglandins regulate haematopoietic stem cells through modulation of Wnt signalling and we will determine if this role is conserved in neural stem cells. We will also address how cell proliferation in the eye and tectum is coordinated. Using mutants and transgenic animals, we will test how retinotectal innervation and activity regulates the production of neurons from the tectal stem cell niche. Finally, we will generate further tools and resources to help establish the larval tectum as a powerful model to study stem cell behaviour in the intact brain.

Who will benefit from this research? In addition to scientists in the field, our work is likely to be of long-term benefit to clinically oriented colleagues and those working in biotech and pharmaceutical industries who lack suitable models for drug screening and other stem-cell related activities. Outside of the academic, clinical and commercial sector, our work will have impact on students in other fields of study including school children, and upon the general public. How will they benefit from this research? As is clear from BBSRC's own statements, work on stem cells is crucial for many aspects of biological science, and most importantly for human wellbeing. Work on stem cells has already immensely influenced medicine and provided the foundation for therapies to replace or repair a variety of organs such as bladder [1], liver [2] and heart [3]. In vivo work on neural stem cells, which could result in regenerative therapies for neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, and nervous system injury, is hindered by the inaccessibility and scarcity of such cells in mammals.Our project will develop a new model system to study neural stem cell biology in vivo. Knowledge gained and tools generated will help other researchers design new drugs and technologies to affect stem cell behaviour, and potentially create cures for diseases that involve nervous tissue damage. For example, a main aim in this project is to label and visualise living stem cells in the zebrafish brain with fluorescent proteins. Other academic, clinical or pharmaceutical/biotech investigators will be able to use this model and the resources we develop to isolate the stem cells and identify the molecular signatures of neural stem cell pluripotency, and/or to screen for molecular compounds that can affect the survival and/or proliferation of neural stem cells. Our experience is that school children and students in non-scientific fields are fascinated when introduced to scientific research in the lab or through presentations. Such exposure can direct career decisions and can inspire creativity in the students' own areas of expertise (4). The public benefits from a better understanding of science and scientific terms and a more intangible appreciation of the beauty of the developing embryo. What will be done to ensure that they benefit from this research? In addition to publishing in scientific journals, we present our work online where anyone can download our publications and read about ongoing projects prior to publication. We write public-friendly reports of all of our publications [4] - these help to ensure that the media understand and accurately report our work and it enables the public to gain an understanding of cutting edge research without having to have a background in science. It also allows researchers in more clinically or pharmaceutically oriented settings to gain an appreciation of how our research could be used to facilitate their own work. We undertake a lot of outreach activity [5], including placements of school kids in the lab, school visits, talks to non-scientific audiences, and web presentations. Our images are widely used in museums, for UCL publicity and other uses. Subject to any MTA agreements from third parties, we share all tools and transgenic fish lines generated during this project with anyone who is interested in using them. Our participation in the UCL Centre for Stem Cell Research, Tissue Engineering and Regenerative Medicine will facilitate the opportunities for clinical collaborations during the project. [1] Atala et al. (2006) Lancet 367(9518):1241-6. [2] http://news.bbc.co.uk/2/hi/health/6101420.stm [3] http://www.northwestern.edu/newscenter/stories/2009/03/cd34stem.html [4] www.ucl.ac.uk/zebrafish-group/outreach/summaries/index.php [5] www.ucl.ac.uk/zebrafish-group/outreach/