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

Lead Research Organisation: University of Aberdeen
Department Name: School of Medical Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description The same important molecular control pathway (called Wnt/beta-catenin signalling) regulates different and specific responses in different tissues and at different stages of development. We have developed a non-hypothesis constrained experimental approach to analyse the molecular mechanistic differences in the accessible early amphibian embryos. We have successfully established ChIP-seq and RNA-seq technology and found that molecular pathway components (beta-catenin) associate with chromatin close to many more genes than are then transcriptionally regulated in the specific stage- and tissue-specific context. We further found that interaction with other molecular pathways, but different pathways for regulation of different genes, is important for specific expression of genes regulated by the same molecular control pathway (Wnt signalling) in the specific tissue at the specific stage of development. This represents a basic science project which ultimately informs about key molecular mechanisms that are important for understanding of birth defects and cancer.
Exploitation Route Our findings have raised important further questions about these widely relevant molecular mechanisms, which our now established experimental set up will be able to address. In particular, it now seems important to understand not only how target genes of this important control pathway (Wnt/beta-catenin signalling) are activated in a specific cellular context (tissue and/or stage specific), but how other established Wnt/beta-catenin target genes can remain silent even though in at least some cases molecular pathway components (beta-catenin) associate with chromatin close to many of those silent Wnt/beta-catenin target genes.

This context-specific response to this conserved key control pathway (Wnt/beta-catenin signalling) is particularly important for normal differentiation of cardiomyocytes (or heart muscle tissue) during normal development and possibly also if therapeutically manipulated for regeneration of injured heart muscle. Early activity of this Wnt pathway promotes eventual cardiomyocyte differentiation but at a later stage activity of this same pathway restricts normal heart muscle formation and regeneration. Thus the context-specific response in the same tissue changes dramatically in a stage-specific way, which has great relevance for normal heart development, birth defects and possibly regenerative medicine. We are currently establishing experimental approaches in amphibian embryos, embryonic explants and human Embryonic Stem Cell culture to study the functional role of this key control pathway (Wnt signalling) in vertebrate and specifically human heart development and cardiomyocyte differentiation. With our molecular and bioinformatics expertise from the current project we plan to investigate the particular molecular control mechanisms mediating these precise, fundamentally different and strictly stage-specific responses during formation of one of the medically most important functional tissues.
Sectors Education,Healthcare,Leisure Activities, including Sports, Recreation and Tourism,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description We have established the experimental system and the analytical molecular tools in 2012 and 2013 and got the results in late 2013 and early 2014. Currently our discoveries have only impacted on academic beneficiaries (we have given specialist seminars at Universities and international and national scientific conferences).
First Year Of Impact 2014
Sector Education
Impact Types Cultural

 
Description BBSRC responsive mode
Amount £396,769 (GBP)
Funding ID BB/M001695/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2015 
End 03/2018
 
Description Speaker at DevCom-ITN conference on Gene-Regulatory Systems in Development in (Parador de Carmona, Spain) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Research seminar to international expert audience.
Year(s) Of Engagement Activity 2017
URL http://www.DevCom-ITN.eu/conference
 
Description Academic Seminar Stefan Hoppler at Weatherall Institute of Molecular Medicine, Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Expert questions and scientific discussion. Beneficial for us thinking about our research project in particular and our research plans in general.

Particular interest in our molecular modelling approaches. I was helping setting up some software.
Year(s) Of Engagement Activity 2014
 
Description CELL DEVELOPMENTAL AND CANCER BIOLOGY SEMINAR (IMS, UoA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Presentation of Research Project to Professional Colleagues.
Year(s) Of Engagement Activity 2015
URL http://www.abdn.ac.uk/ims/
 
Description EMBO Workshop on Wnt signalling (Stefan Hoppler) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Expert feedback.

Maybe too early to say
Year(s) Of Engagement Activity 2014
URL http://wnt2014.mtci.com.au
 
Description Gordon Research Conference Wnt Signaling, Mount Snow, VT 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact International meeting of experts in the Wnt signaling pathway.
Year(s) Of Engagement Activity 2019
URL https://www.grc.org/wnt-signaling-conference/2019/
 
Description Invited research seminar at Department of Biology, University of York 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact University research seminar, discussion of potential future collaborations.
Year(s) Of Engagement Activity 2017
URL https://www.york.ac.uk/biology/news-events/seminars/cellmolecularstructuralbiology/profstefanhoppler...
 
Description Invited speaker at 16th International Xenopus Conference (Crete, August/September 2016) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Important international meeting happening every other year bringing together researchers using Xenopus and other amphibians mainly for fundamental biomedical and biological research. Our invited research seminar sparked many interesting questions and discussions.
Year(s) Of Engagement Activity 2016
URL http://www.xenopus16.com/17789
 
Description Poster Presentation at Joint BSBD/Nordic Autumn Meeting (Stockholm, 25-27 Oct. 2017) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Expert feedback on our work.
Year(s) Of Engagement Activity 2017
URL https://www.med.lu.se/bsdbnordic2017conference
 
Description Research seminar at Okinawa Institute of Science and Technology (25 April 2018) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Research Presentation to Professional Audience
Year(s) Of Engagement Activity 2018
 
Description Scottish Developmental Biology Group Meeting (Dundee, 3 June 2015) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Dr Yukio Nakamura presented a research seminar about " Context-specific Wnt target gene expression is controlled subsequently to beta-catenin recruitment to genetic loci"
Year(s) Of Engagement Activity 2015
 
Description UK Xenopus Meeting 2014 (Dr Yukio Nakamura) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation paper presentation
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of our unpublished results produced many expert questions and subsequently discussion of the implication. It helped us refine our subsequent experimental approach.

Improvement of our experimental design. Inspired others in the Xenopus field to apply similar experimental approaches and analysis methods to their scientific and biomedically relevant research questions.
Year(s) Of Engagement Activity 2014
URL http://www.kcl.ac.uk/dentistry/newsevents/events/eventsrecords/UK-Xenopus-Meeting-2014.aspx