DIVERSIFICATION OF VERTEBRATE T-CELL FACTOR (TCF) STRUCTURE AND FUNCTION IN EVOLUTION AND DEVELOPMENT

"Nothing in biology makes any sense; except in the light of evolution" is the famous quote by the eminent geneticist Theodosius Dobzhansky about the understanding of biological mechanisms.

Here we propose to follow his advice in order to gain unique, novel and fundamental insight into the function of a prominent biological mechanism, called Wnt signalling, which normally regulates embryo formation and regeneration in humans and other animals. We also know that Wnt signalling can contribute to human disease, such as cancer and diabetes, when it is defective or is abnormally activated.

Wnt signalling is a molecular pathway that regulates where and when specific genes are switched on during the building of the embryo, and during maintenance and regeneration of some adult body organs, but unfortunately also in human disease. At the end of the Wnt pathway is a molecule called TCF. TCF contacts and controls the 'switch' DNA sequences in genes that are regulated by Wnt signalling; TCF switches these genes on, when Wnt signalling is active and switches them off, where Wnt signalling is silent.

In animals without a backbone (invertebrates) there is just one TCF, so that the Wnt-TCF pathway is relatively straightforward. In humans and closely related animals with backbones (vertebrates) there are many TCFs. Here we will investigate whether having several different TCFs enables Wnt signalling to carry out much more diverse roles to help build the bodies of more complex vertebrates and humans. Knowing how the different TCF proteins have different effects on the genes that they control in the embryo is also highly likely to improve our understanding of how they do different things in various diseases, like cancer and diabetes.

In this project we will combine information from several vertebrates and closely related invertebrates on the separate genes that make these different TCFs. We will examine and dissect the different and unique roles of the distinct vertebrate TCFs. Because of the prominent role of Wnt signalling in human disease, such as bowel and liver cancer and Type-2 diabetes, many biomedical researchers are interested in studying the role of the Wnt pathway in normal biology and in these diseases, including specifically the role of TCF molecules. However, adopting the powerful comparative approach, as we will do in this project, has not yet been tried. Our approach will provide us with unique novel insights into this important biomedical mechanism for normal embryo formation, and ultimately also human disease.

We aim to provide insight into the origins and molecular bases for the diversity of TCF functions downstream of Wnt signalling in vertebrate development. Wnt/beta catenin/TCF signalling mediates diverse roles in embryonic development, stem-cell-mediated regeneration and human disease. While the upstream Wnt/beta-catenin pathway is remarkably conserved; the diversity of functions in vertebrates clearly correlates with increased diversity of TCF transcription factors expressed from vertebrate genomes. In this interdisciplinary collaboration we will investigate how this step change in TCF diversity contributes to diversification of Wnt function in vertebrates. We will test the hypothesis that whole-genome duplications led to diversification of vertebrate TCF genes and possibly increased isoform-coding, which then enabled acquisition of redundancy, as well as, segregated (sub-functionalisation) and novel functions (neo-functionalisation) in vertebrates.

We will particularly investigate whether the vertebrate TCF7E isoform retains functions of the invertebrate TCF and explore whether the TCF diversity is particularly important for embryonic development of the vertebrate CNS. We will meticulously compare gene structure and synteny of TCF genes in genomes of different vertebrates with those of the most closely related invertebrates, the chordates Ciona and amphioxus. We will determine the embryonic expression of TCF genes and isoforms, and assay their function in the Xenopus vertebrate model and in the chordate invertebrate Ciona. We will be able to carry out exciting cross-species TCF swap experiments to assess the extent to which ancestral functions are retained in vertebrate TCF orthologs and in vertebrate embryos. This project will produce a comprehensive understanding of the origin of the diversity of vertebrate TCF structure and the functional impacts in vertebrates, with far-reaching implications for the understanding of Wnt signalling in human disease biology.

This project represents fundamental research with immediate impact for researchers, intermediate impact on public understanding, and potential for future impact on biotechnology and health services.

IMPACT ON SCIENTIFICALLY SKILLED WORKFORCE: There is currently still a considerable paucity of trained researchers at the interface between bioinformatics and experimental developmental biology in the UK. The post-doctoral researchers on this project will directly benefit from career development and skills training in this interdisciplinary and collaborative research environment. The St Andrews post-doc will start with bioinformatics but then apply some of the bioinformatics discoveries into laboratory developmental biology experiments; while the Aberdeen post-doc will focus on functional experiments in the laboratory that are informed by bioinformatics analysis.

PUBLIC UNDERSTANDING OF SCIENCE IMPACT: The general public are also interested in both the evolution of biological diversity on this planet and the causes of human disease. Impact on the general public will be pursued through press releases from our University press offices. We will also expand the Tcf/Lef family Wikipedia web-page, since this is often the first port-of-call for most people when trying to find out about a topic. This Wikipedia page is currently very sparse. We will also contribute to public outreach and widening access events, with a display of amphioxus and Ciona adults alongside Xenopus tadpoles, with posters and scientists on hand to discuss use of these animals in a comparative fashion to help understand human biology and disease. School visits to our Universities are another avenue for us to introduce our study species to school children and explain the concept of model systems to study medically relevant biology. We will also run a student summer internship program at St Andrews. We will also extend David Ferrier's existing link to the St Andrews public aquarium (60,000 visitors per year), producing a display of Ciona and Xenopus alongside a poster explaining their usefulness in studying fundamental biology relevant to human development and disease. A similar exhibit will also be produced in the new public outreach space in the redeveloped Gatty Laboratory, which faces onto the Fife coastal path and East Sands beach, with over 55,000 users of the coastal path alone each year.

IMPACT ON FUTURE RESEARCH: This research project will have direct impact on the two collaborating research groups and their Universities. This project provides the strong foundation for addressing further research questions with future investigations of specific functions of vertebrate TCF genes and of the regulatory mechanisms controlling alternative isoform expression, such as tissue-specific alternative splicing regulators. We are also keen to expand our analysis of TCF isoform expression and function to cancer genomes and gene expression data from cancer biopsy samples.

IMPACT ON BIOTECHNOLOGY: Our connection with Nanopore will improve application of their technology for studying alternative mRNA expression, which will impact beyond TCF and Wnt signalling on the study of the still greatly neglected role of alternative splicing in vertebrate development. As a follow-up to this project, we plan the development of isoform-specific antibody tools with biotech companies. Such isoform-specific antibody tools could eventually become useful for diagnosis of specific subtypes of particularly colorectal cancer, since expression of different TCF proteins and possibly isoforms is thought to influence cancer progression. Furthermore, SNPs strongly associated in GWAS studies with increased predisposition for type-2 diabetes map to within the TCF7L2 gene, and may be caused by abnormal tissue-specific expression of TCF7L2 isoforms. Isoform-specific antibodies will be impartant research tools and could eventually be of diagnostic value.