Context-specific regulation of Wnt/beta-catenin target genes

GENERAL RESEARCH QUESTION: One of the most important questions in biology asks how our body is built. The many cells in the embryo communicate with each other, first to arrange the general body plan and then to regulate formation of specialised cells to build our organs. These embryonic cells communicate with each other using molecular cell-to-cell signalling mechanisms. This process persists after birth when organs are repaired and tissues regenerated by continued formation of such specialised cells from resident adult stem cells. The same cell-to-cell signalling pathways as in the embryo regulate this process in the adult. Defects in signalling mechanisms consequently do not only lead to birth defects in babies but also to diseases in adults, such as cancer.

SPECIFIC RESEARCH QUESTION: Wnt signalling is one of our most important molecular cell-to-cell signalling mechanisms. We already have a good understanding of the linear molecular Wnt signalling cascade, which functions to switch on or off specific genes that are needed to build specialised cells in our functional organs. However, we do not yet understand how Wnt signalling manages to find the correct genes to switch on or off in different tissues and organs. We clearly need to uncover these important mechanisms that allow Wnt signalling to function repeatedly during embryonic development and in different stem cells, while nonetheless reliably switching on or off the correct set of genes in each of these types of cells.

EXPERIMENTAL SYSTEM: We have recently taken advantage of state-of-the-art molecular analysis protocols, which allow us to identify in a comprehensive manner all the genes that are switched on by Wnt signalling in a particular group of cells. We have established these methods in an accessible experimental system where we knew already about a dramatic change happening in a short time in the way Wnt signalling regulates early embryonic cells compared to slightly older cells. With these new methods we have now uncovered that Wnt signalling indeed switches on very different genes in later cells than the few we already knew about in early cells.

SPECIFIC OBJECTIVES:
1. Since we are now getting to know very well the set of genes switched on in later cells, we now want to carry out a proper comparison with an equally well analysed set of genes switched on in earlier cells.
2. Because we know generally which group of molecules touch switches as they are turned on or off, we now want to investigate whether different individual molecules touch different switches in different tissues, which may then allow different sets of genes to be switched on in early as compared to later cells.
3. When we know the particular switches in early and in later cells and the molecules touching these switches in early and later cells, we will investigate the molecular mechanisms by which these molecules are guided to the correct switches.

OUTCOMES AND BENEFITS: Our experimental model system allows us now to address these scientific questions. However, the findings from our experiments are going to be relevant not just for this experimental model system. The same Wnt signalling cascade also controls stem cells in the adult and for instance cancer cells in disease. A wide range of scientific fields will therefore benefit from our results about how different specific genes can be switched on or off by Wnt signalling in different tissues.

Our experiments will also develop molecular tools and experimental approaches that will prove useful to others as they need to identify all the genes that are switched on by a certain molecular pathway in a particular group of cells; or how a specific group of molecules finds, touches and turns switches to activate these genes.

The Wnt/beta-catenin signalling pathway is a conserved cell signalling mechanism that operates during embryogenesis, and in stem cells and cancer. While the fundamental signal transduction mechanism of Wnt signalling has been extensively studied, what is critically missing is a good understanding of how the same Wnt signalling signal transduction elicits context-specific responses in different cell lineages or tissues during different biological processes.

This fundamental question can presently be addressed with early Xenopus embryos, where a radical change in the developmental response to Wnt signalling (maternal versus zygotic Wnt signalling) happens in a short time.

We have previously demonstrated that the context-specific response to Wnt signalling is regulated by different TCF/LEF transcription factors. With a complementary approach employing RNA-seq, we have recently identified novel zygotic Wnt signalling targets; but known maternal targets remained refractory, suggesting robust mechanisms that specifically induce context-specific Wnt targets and exclude others. We have also found that their context-specific expression required combinatorial signalling (BMP pathway). Additionally, we have established beta-catenin ChIP protocols that enable us to investigate more precisely context-specific regulatory mechanisms of Wnt signalling.

In order to understand these robust mechanisms we will comprehensively identify maternal Wnt targets, test whether zygotic Wnt targets are also refractory to maternal Wnt signalling, and clarify whether the robust mechanisms driving context-specific response rely on the function of different TCF/LEF transcription factors or on the molecular interactions between TCF/LEF and different transcription factors in combinatorial signalling.

We can now address this fundamental question in Xenopus, but our findings will be relevant for medically relevant tissues where this question is currently more difficult to study.

INTRODUCTION: This is a basic science project; it addresses fundamental questions about conserved molecular biology mechanisms, which are important for embryonic development, stem cell biology and human and animal diseases, such as particularly cancer. This application has therefore some immediate impacts but also many indirect impacts.

IMPACT FOR HUMAN (AND ANIMAL) HEALTH AND APPLIED TRANSLATIONAL RESEARCH: Wnt signalling is 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 (e.g. SERKAL syndrome in the kidney) and cancer (particularly colorectal and liver cancer). Understanding the molecular mechanisms that regulate the specificity of the transcriptional and cellular response in different tissues is therefore fundamentally important for development of therapeutic strategies aimed at treatment of developmental abnormalities and cancer progression. Furthermore, Wnt signalling is important for development and implementation of stem cell-based therapies in regenerative medicine, particularly for cardiomyocyte (heart muscle regeneration).

IMPACT ON GENERATION OF A SCIENTIFICALLY LITERATE WORKFORCE: This project will train the next generation of biomedical researchers not only directly by supporting Dr Nakamura's research career but also indirectly by contributing to a research-led environment for teaching of postgraduate and undergraduate students.

IMPACT ON WIDER PUBLIC: Members of the public are interested in scientific progress and health issues. Increased understanding of gene regulatory mechanisms of the medically-relevant Wnt signalling pathway will contribute to public understanding of important healthcare issues, which will not only inform public opinion, but also feed into national and international public health policy decision making.

IMPACT ON PHARMA AND BIOTECH INDUSTRY: Wnt signalling, and particularly context-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 context-specific mechanisms as proposed for this project will increase the knowledge base needed for continued development of more sophisticated Wnt pathway targeting drugs.

CONCLUSION: This study will therefore directly and indirectly contribute to the health quality and ultimately the economic wealth of life in the UK.