An evolutionarily restricted role for purinergic signalling in the maintenance of retinal stem cells

During embryogenesis the eye is formed by a highly organized and stereotyped pattern of events, in which retinal progenitor cells divide (proliferate) many times before exiting the cell cycle and forming the different neuronal cell types of the adult retina in a process called differentiation. Once formed, the mammalian eye is thought to be incapable of regeneration. Thus, any loss of retinal cells, including the light-sensitive photoreceptors, either by disease or injury during life is permanent. In contrast, the eye of lower vertebrates, such as fish and frogs, continues to grow throughout the life of the animal. New retinal neurons, including photoreceptors, are produced by the proliferation of a population of stem cells that reside in a region called the ciliary marginal zone (CMZ), at the very periphery of the retina. Over the past decade, there has been significant interest in the possibility of retinal repair in humans following the discovery of a population of cells within the ciliary epithelium (CE), found within the ciliary body of the mammalian eye, a region analogous to the lower vertebrate CMZ. These cells exhibit a number of stem cell-like properties, although it is not yet clear if these cells can go on to form all the cell types of the retina. More importantly, these cells only proliferate when cultured in a dish - they are dormant in the eye itself. This raises the question of whether these cells represent a population of stem cells that are similar to those found in frogs and fish but that have lost the capacity for proliferation and repair during evolution. Furthermore, it is conceivable that these cells could be reactivated to generate new retinal neurons. The mechanisms that regulate both the proliferation of the stem cells in the lower vertebrate and the apparent dormancy of this stem cell-like population in mammals are poorly understood. Recently, one signalling pathway, called purinergic signalling, has been shown to be essential for turning on a number of genes required for eye formation. We have also shown that this signalling mechanism is very important for controlling the proliferation of retinal progenitor cells in the early stages of eye development in the embryo. Here, we aim to determine whether or not purinergic signalling represents a fundamental regulatory mechanism that controls the proliferation of retinal stem cells at the ciliary margin. Moreover, we wish to determine whether or not this signalling system has become lost or restricted during evolution and so provide an explanation for why the stem cells of the mammalian CE no longer proliferate. We have already found that purinergic signalling is involved in the proliferation of mammalian retinal stem cells in the culture dish. We have also found that a particular type of receptor that is critical for purinergic-mediated proliferation elsewhere in the brain is not present in the mammalian CE, but is found in these cells when they are cultured in the dish. This means it may be possible to induce the normally dormant stem cells of the mammalian eye to proliferate and form new retinal neurons in the eye. Therefore, we aim to firstly examine the role of purinergic signalling in controlling retinal stem cell proliferation both in the adult eye and in the culture dish. We will also determine the downstream pathway of purinergic signalling that leads to this proliferation. We will compare evolutionarily distinct species to examine the role of purinergic signalling in the retina of these animals and finally we will determine whether or not it is possible to reactivate the stem cells of the mammalian CE by purinergic signalling. Although only theoretical at this point, it is possible that the last objective may provide information for the repair of the retina damaged through disease or injury.

The lower vertebrate eye continues to grow throughout the life of the organism. New neurons are produced from a region called the ciliary marginal zone (CMZ), which represents a stem cell niche. By contrast, the adult mammalian eye ceases retinal neuron production once development is complete and any loss of retinal cells during life is permanent. A recently identified population of progenitor cells, termed retinal stem cells (RSCs), located in the ciliary epithelium (CE), part of a structure analogous to the CMZ, exhibits stem cell-like properties in culture but is quiescent in the adult eye. Purine nucleotides, acting via purinergic receptors, have been shown to control the proliferation of retinal progenitors in development and of other stem cell populations elsewhere in the adult CNS. Here, we aim to determine whether or not purinergic signalling represents a fundamental regulatory mechanism for controlling the proliferation of adult RSCs. Moreover, we wish to examine whether this pathway has become lost through evolution and if its absence provides an explanation for the apparent dormancy of the RSCs found in the adult mammalian CE. We will use a combination of molecular and cell biology techniques including neurosphere cell culture, immunocyto- and immunohisto-chemistry, in situ hybridization, qRT-PCR, confocal microscopy, live cell imaging and in vivo ocular gene transfer. These experiments aim to; confirm preliminary findings demonstrating a role for purinergic signalling in the promotion and maintenance of mammalian RSC proliferation in culture; examine whether it is possible re-activate the quiescent mammalian RSCs in vivo by ectopic expression of purinergic receptors and related enzymes; examine the role of purinergic signalling in controlling RSC proliferation in lower vertebrates (chick and Xenopus) and examine whether or not purinergic signalling represents an evolutionarily restricted mechanism for the maintenance of RSC proliferation.

There is considerable excitement regarding the potential of stem cells in the treatment of disease, in the international research community, in government and in the wider public. It is becoming clear, however, that these cells are very complex, with significant differences between the various populations of cells with stem like properties in the adult organism, compared with those derived from embryonic sources. It is essential therefore to understand the regulatory mechanisms controlling these potentially powerful cells. By understanding the biological context within which different stem cell populations are found, we should learn much that can be applied to, for example, the in vitro expansion and directed differentiation of these cells into specific cell types, such as spinal cord neurons, heart muscle, retinal neurons etc. This project will examine the properties of a population of stem-like cells within the adult mammalian eye. As yet, these cells are of unknown function and do not contribute to retinal repair in the face of disease or injury, but they do exhibit stem cell properties in vitro. By comparing species from different points on the evolutionary scale it is hoped that we will gain insights into why lower vertebrates are capable of repairing their retina, while mammals are not. While this project does not have a therapeutic goal, it is conceivable that the signalling mechanism under investigation could re-activate the otherwise quiescent population of stem cells in the mammalian retina and lead to appropriate regeneration of the neural retina. If so, it could lead to novel strategies for the repair of retinal degeneration. This would clearly have significant impact on the health and quality of life of sufferers of retinal degenerations such as retinitis pigmentosa, which currently represent the leading cause of blindness in the the developed world. Moreover, the number affected by such disorders is expected to rise with an increasingly ageing population. We anticipate the following groups being beneficiaries from the proposed research; researchers in retinal development, stem cell biology and/or neuronal regeneration; researchers interested in gene transfer as a method for ectopic expression of genes within the eye; clinicians with an interest in neuronal repair and regeneration; advisory boards involved in the funding of stem cell research. While we do not anticipate this work have patentable outcomes, the Institute of Ophthalmology has considerable experience in filing patents and we will seek to exploit any new intellectual property rights that might arise. We anticipate publishing data arising from this study during the lifetime of the grant. We regularly present our findings at international cross-disciplinary conferences including the major international conferences in Neuroscience, Vision Research, Gene Therapy and Stem Cell Research. This ensures engagement with a broad spectrum of researchers in related and non-related fields and a chance to share ideas and establish collaborations. It also means that there will be a timely dissemination of the findings arising from this study. Any significant new scientific discoveries will be communicated, as appropriate, through the Press Offices at UCL and the BBSRC. We have considerable experience in presenting research to journalists and the public. Recent studies by the applicants, published in Nature and NEJM, were featured on TV and radio and in broadsheet and tabloid press. We regularly present our work on cell and gene therapy to lay audiences from both patient and government organisations. With regard to the named researcher, this project will furnish her with considerable expertise in a wide range of molecular and cell biology techniques. Together with her existing skills base and the likely publications arising from this work, this will place her in an excellent position to gain a post-doctoral position of her choosing.