Stem cell fate: exploiting the Drosophila germline to unravel the role of a conserved translation repression complex

Stem cells are different from other types of cells as they can divide to renew themselves (ie remain as a stem cell) and simultaneously differentiate into specialised cells with distinct identities. For example, embryonic stem cells can form all of the different cells, tissues and organs during development, whereas there is evidence that germline stem cells can give rise to functional eggs and sperm. Given this unique property, stem cells have the potential to revolutionise medicine by providing both new treatments for diseases such as neurodegenerative and fertility disorders, and novel strategies for repair of damaged tissue, including those associated with heart disease and spinal injury. One of the primary hurdles to be overcome is the induction of stem cell differentiation into multiple different cell types within the laboratory. This difficulty arises because the mechanisms underlying a stem cell's decision to renew itself or differentiate are not fully understood. The research in this proposal aims to understand one of the mechanisms that controls this decision.

One of the premier models for stem cell research is fruitfly germline stem cells, as the proteins which are important for development of the fruitfly are the same as those which control development in humans. In addition, the fruitfly is amenable to a whole range of experimental approaches on a rapid timescale, making it a commonly used research model, and one that is a less morally contentious alternative to animal models. We have recently used fruitfly germline stem cells as a model to investigate the basis of the decision by which stem cells choose to either remain a stem cell or differentiate into specialised cells. Our work has focused on two proteins, called Pumilio and Nanos, which function to repress translation (ie the synthesis of proteins) in germline stem cells. We have identified a specific mRNA that is repressed by Pumilio and Nanos in germline stem cells, and shown that loss of repression of this mRNA causes the stem cell to differentiate rather than renew itself. Interestingly, the protein encoded by the mRNA has, in a separate study, recently been shown to cause mouse stem cells to differentiate. This result demonstrates that fruitfly stem cells are a powerful model for identifying factors important for promoting differentiation of mouse and human stem cells.

The aim of this proposal is to extend our study of the translation repressors which function in fruitfly germline stem cells. We will identify other mRNAs that are repressed by Pumilio and Nanos, and investigate how these mRNAs are selected. We will then test how loss of this repression in a germline stem cell influences its decision to either renew itself or differentiate. Pumilio and Nanos also have important roles in controlling the stem cell fate decision in human stem cells. Therefore, it is anticipated that results from our study will be relevant to other types of human stem cells and in the longer term be useful for promoting stem cell differentiation into specific cell types within the laboratory setting, in order that the full therapeutic potential of stem cells can be harnessed.

The Puf family of translational repressors function in stem cell maintenance. Their role is so deeply conserved, ranging from planarian to human stem cells, that it has been suggested to be the ancestral function of Puf proteins. However, the precise molecular basis by which Puf proteins maintain stem cells is unknown. In the Drosophila ovary, germline stem cells (GSCs) divide to form a daughter that is maintained as a GSC, and another that differentiates. In this system, translational control is a critical determinant of the GSC fate, with the Pum and Nos translational repressors required for self-renewal. The global aim of this proposal is to determine the molecular mechanisms by which the Pum-Nos translation repression complex maintains Drosophila GSCs. To this end, it is proposed to identify the precise targeting mechanism by which the Pum-Nos complex represses translation. We will also identify genome-wide mRNA targets of Pum-Nos repression, and test whether miRNAs are involved. These data will be integrated and validated in vivo, thus providing a step change advance in our understanding of the molecular basis of the stem cell fate decision. The Drosophila ovary provides a unique opportunity to address these questions as it is amenable to genetic and transgenic manipulation, vast numbers of GSCs can be easily purified, and in vivo validation is achievable on a rapid timescale. Moreover, this work will build on our recent data identifying the first mRNA target of Pum-Nos translation repression in GSCs. Given the conserved role of Puf and Nos proteins in other stem cell systems, data from this proposal will provide a framework for understanding how translation regulation impacts upon stem cell fate choices in other organisms, including humans, where there is vast therapeutic potential to be harnessed through the precise manipulation of stem cell fate.

Academic beneficiaries: As described in the 'Academic beneficiaries' section, these will include UK and international researchers in the following biological science fields: basic stem cell research, post-transcriptional control of gene expression, cell biology, developmental biology and neuroscience. Academic beneficiaries from other disciplines will include medical researchers developing novel strategies for promoting stem cell differentiation, as well as those generating stem cell models of various diseases in order to study disease progression. Finally, mathematical modelers developing models of stem cell behaviour will also benefit from our research.

General public: In terms of the wider public beneficiaries, the long term impact of this project will be the restorative applications of stem cells to a range of diseases. To name a few, these include Parkinson's and Alzheimer's diseases, heart disease, spinal cord injury, diabetes and arthritis. Even limited medical advances in the treatment of many of these diseases will lead to a huge benefit for society, e.g. a medical advance sufficient to delay the onset of dementia by 5 years is predicted to reduce deaths directly attributable to dementia by 30,000 per annum. Also, the current treatment for many diseases, such as heart disease, involves a transplant. As the existing demand for organs far outstrips those available, the use of stem cells as an alternative to repair damaged tissue would remove the reliance on donated organs.
By deepening understanding of the mechanisms surrounding the regulation of translation and RNA biology, this proposal may also have more far-reaching medical consequences. For example, the defense against viral infection, iron homeostasis, severe diabetus mellitus, arsenite toxicity, oxidative stress and nutrient deprivation all rely upon post-transcriptional control mechansims. Indeed drugs such as Clotrimazole, which inhibit translation via effects on calcium homeostasis, may prove useful as anti-tumour agents. In addition, the drug Rapamycin, which targets translation initiation, is in clinical trials as a treatment for transplant rejection and as a chemotherapeutic agent. The Puf proteins themselves are also clinically important. As Leishmania Puf proteins induce an immune response during parasitic infection, the Puf proteins have been suggested as targets for diagnosing and vaccinating against Leishmaniases which affect >1m people/year. Therefore, long-term beneficiaries of this project will be individual patients who receive treatments that rely upon the direct application of stem cell biology and the understanding garnered from studies such as this in post-transcriptional control.

Public sector: In terms of the public sector, stem cell based therapies have the potential to transform the NHS. The current financial cost of dementia to the UK is over £20 billion per annum, with a quarter of NHS beds occupied by someone with dementia. Therefore, there are enormous economic benefits to be gained in the long term by developing new treatments for dementia and other diseases.

Industry: The ability to precisely manipulate stem cell differentiation is being exploited by the commercial sector for drug discovery and development. 90% of drugs in clinical trials are not approved, despite the process from drug identification to market approval estimated to cost £700m. Reasons for drug failure include toxicity and insufficient efficacy. A new avenue of drug development, which adheres to the 3R principle, involves differentiating stem cells into a particular cell type to then screen drug compounds more effectively. This requires pure cell populations so that results from different compounds can be accurately cross-compared. Therefore, strategies for inducing differentiation of stem cells into a pure population of a given cell type will benefit the drug development process, where only a 10% improvement is estimated to save £70m.