Tissue-specific regulation of gene expression by Wnt/beta-catenin signalling

One of the most important questions in biology is to explain how our body is built during embryogenesis. The many cells in the embryo communicate with each other (cell-to-cell signalling), initially to arrange the orientation of the general body plan and eventually to regulate formation of specialised cells (cell differentiation). This process does not end at birth, since many organs need to be repaired and tissues regenerated in the adult by continued formation of such specialised cells. The same cell-to-cell signalling pathways regulate this process in the adult as in the embryo. Defects in these signalling mechanisms lead to birth defects in embryos and in adults to diseases such as cancer. We already have a fair understanding of the individual linear cell-to-cell signalling mechanisms. They function to switch on or switch off genes, which issue instructions (called RNA) to build particular components that different cells need to form functional organs. These RNA instructions can be detected experimentally, but using conventional technology we can only look for specific RNA instructions. New state-of-the-art deep-sequencing methods allow the possibility of reading all the RNA instructions issued in a particular tissue. There is a fundamental gap in our current understanding of how individual cell-to-cell signalling mechanisms can so precisely control the exact combinations of RNA instructions needed in different cell types. How are specific genes switched on in particular cells? There are many different cell types but only few cell-to-cell signalling mechanisms, so the inputs from different cell-to-cell signalling mechanisms may be combined to produce the appropriate response. This integration could happen in several different ways, but the combination of switches regulating individual genes is obviously the most likely level for control. We will use new sequencing technology to read all the RNA instructions issued in a tissue to identify potentially regulated genes. We will confirm these candidate genes by verifying whether they are regulated by cell-to-cell signalling mechanisms in the correct tissue. Once confirmed, we will examine them for combination of switches through which these genes are regulated. We will be able to compare the whole complement of these genes in order to recognise whether they are mostly regulated by the same mechanisms, or whether different mechanisms apply to different genes. The insight gained will allow us to propose and then test possible molecular mechanisms that integrate cell-to-cell signalling to regulate genes in a specific tissue. Understanding how signalling mechanisms regulate genes will be relevant to understanding human and animal embryos, adult stem cells and cancer. However, this important issue is difficult to study directly in many of those systems. Xenopus embryos are ideal for tackling this fundamental question: they are accessible for experimental analysis; the large embryos provide sufficient material for sequence analysis; and we and others have intensively studied developmental processes in the early Xenopus embryo to suggest that at least one of the involved mechanisms involves integration of WNT and BMP signalling. Both of these signalling pathways are highly conserved between Xenopus and humans, and are important for embryogenesis, stem cells and cancer. Our pilot experiments show that our experimental approach is feasible and support our working hypothesis that combinatorial Wnt and BMP signalling regulates tissue-specific genes.

There is a fundamental gap in our understanding of how the conserved Wnt/beta-catenin pathway conveys distinct yet specific instructions in different tissues. The early Xenopus embryo provides an experimentally tractable model system to address this fundamental question. There is a dramatic shift in response to Wnt signalling from the dorsal-promoting effect of maternal Wnt signalling to the ventral-promoting effect of zygotic Wnt signalling. We previously demonstrated that stage-specific Wnt regulation is mediated by different Tcf/Lef transcription factors. However, such a top-down experimental approach by itself is limiting. The recent revolution in sequencing technology now enables a novel complementary bottom-up approach: 1) We will use SAGE/Solexa-sequencing to identify target genes specifically activated by zygotic Wnt signalling in experimentally ventralised tissue (using established inducible tools and tested experimental approaches to restrict the analysis to directly regulated Wnt target genes). 2) We will validate candidate genes by testing whether they are normally expressed in ventral mesoderm and whether zygotic Wnt signalling is required for their expression. We will use bio-informatics analysis to identify potential regulatory elements and test to what extent they mediate tissue-specific Wnt regulation (using reporter gene assays and Xenopus transient transgenics). 3) We will investigate in vitro molecular interactions among signalling components and with identified regulatory DNA sequences to discover molecular mechanisms of tissue-specific Wnt regulation. We will be able to test directly the importance of any molecular mechanisms for development using in vivo experiments in Xenopus embryos. Our Pilot experiments have already identified vent1 as a direct, tissue-specific Wnt target gene and suggest that combinatorial Wnt and BMP signalling contributes to tissue-specific gene regulation.

As a basic science project, it has immediate impacts but also indirect impacts. IMPACT FOR HUMAN (AND ANIMAL) HEALTH AND APPLIED TRANSLATIONAL RESEARCH: Wnt signalling provides a major biological mechanism for cell-to-cell communication in humans and animals. Deregulated Wnt signalling can have devastating effects in the human population, contributing to developmental abnormalities and cancer. Since Wnt signalling is repeatedly used, the mechanisms that regulate the specificity of the cellular response in different tissues are fundamentally important for development of therapeutic strategies aimed at treatment of developmental abnormalities and cancer progression. Furthermore, Wnt signalling will be important for development and implementation of stem cell-based therapies aimed at diseases such as diabetes, brain disorders and other transplant therapies. Although this impact is currently less immediate, we have established collaborations with academic partners in applied and translational areas to make our research more immediately beneficial to human health (see Impact Plan). IMPACT ON GENERATION OF A SCIENTIFICALLY LITERATE WORKFORCE: This project will train the next generation of biomedical researchers not only directly by training the RF employed but also indirectly by contributing to a research-led environment for teaching of postgraduate and undergraduate students. This impact is more immediate and benefits will be directly achieved through academic supervision, seminars and workshops; and through nurturing the international collaborations. IMPACT ON WIDER PUBLIC: Members of the public are interested in science and health issues. Increased understanding of how regulation of gene expression, embryonic development and diseases such as cancer are linked will contribute to public understanding of important healthcare issues, which will inform public opinion and contribute to national and international policy decisions. This impact is ongoing but by its nature less project-specific. I already play a part in promoting public understanding of science as a committee member of the British Society for Developmental Biology. A dedicated communication team at the University of Aberdeen also communicates key research findings that may be of general interest to the general public via press releases to both the local and national media. Additionally, all research papers are publicised on our institutional website. However, we will also provide more direct communication with the public by participation in public engagement with science events in Aberdeen and by continuing to contribute to the open days of the Institute of Medical Sciences. IMPACT ON PHARMA AND BIOTECH INDUSTRY: Wnt signalling and particularly tissue-specific Wnt signalling mechanisms are important targets for drug development and so one key beneficiary of this work will ultimately be the pharmaceutical and biotechnology companies. Detailed understanding of tissue-specific mechanisms as proposed for this project will increase the knowledge base needed for continued development of more sophisticated Wnt pathway targeting drugs. This impact is not immediate but benefits might be realised soon after the publication of our findings in peer-reviewed journals or presentations at specialised scientific meetings. Additionally, the Research and Innovation Unit at the University of Aberdeen have extensive experience in identifying research with potential for commercial exploitation (and IP protection). They also have multiple contacts with biotechnology and pharmaceutical companies, which will be directly exploited if our findings are assessed as having any commercial potential. We have also established collaborations with academic researchers directly involved in drug development to make our research more directly beneficial for drug development. This study will therefore directly and indirectly contribute to the economic and health quality of life in the UK.