Quantitative analysis of cytoneme-based Wnt trafficking and signalling in vivo

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

Cell-to-cell communication is essential for regulation of development of all multicellular organisms. Intercellular communication is based on chemical stimuli - including signalling proteins - which regulate the cellular behaviour in a tissue.
An important family of signalling proteins that orchestrate development is the Wnt signalling family. Wnts regulate vital cellular processes including how fast cells divide; the fate of cells or how to differentiate into different forms; and how cells move. Wnt signalling is therefore fundamental to the development of early life (e.g. embryogenesis), organ development, wound healing, and regeneration. We know that a relatively small and specific group of cells control and distribute Wnt proteins controlling development. Adjacent, larger groups of cells then respond to the signal. Wnt function is therefore dependent on precise delivery of Wnt proteins from producing cells to target cells.
Currently, how Wnt proteins are transported between cells to activate signalling is unknown. As such we do not understand how the message is delivered from one cell to another. This proposal aims to understand, for the first time, how the message is conveyed between cells. In preparation for this proposal, the lead scientist has revealed the existence of a completely unexpected cell-to-cell transport mechanism for Wnt proteins. Specific finger-like cell membrane protrusions - called cytonemes - carry Wnt proteins to their tips and transport them to neighbouring cells. After contact with the target cell Wnt proteins are taken up by the responding cells. This process leads to signal activation in a target cell. Impairment of the number of Wnt protein transported on these signal protrusions leads to severe consequences during development, leading to malformation of tissues and severe developmental difficulties.
Understanding the systems that govern this newly identified transport system is therefore fundamental for understanding how Wnt functions to elucidate Wnt signal function during embryogenesis and tissue homeostasis. This knowledge will provide the foundations to be able to manipulate Wnt protein transport to control the activity of Wnt signalling cascades in regeneration and diseases. Based on our preliminary work, we propose that the Wnt proteins control their own transport mechanisms: We hypothesize that Wnt triggers a specific set of receptors, which activates formation of cytonemes, and the amount of Wnt signals handed over from these "signalling cell fingers" to the target cell is crucial for the level of signal activation. We will use our established, state-of-the-art, genetic strategies, combined with advanced imaging techniques pioneered by the research team to measure the amount of Wnt protein transported in a living zebrafish embryo. This will for the first time allows us to understand how this signalling operates.
By the end of this project, we will determine how Wnt-producing cells control the emergence of these signalling protrusions. We will further have identified how Wnt signalling proteins traffic from the protrusions to the receptor of the target cell to initiate reciprocal signalling. Furthermore, super-resolution imaging experiments will allow us to quantify Wnt signalling components at the signalling sites and thus understand the actual mechanism of Wnt signalling.
We believe that these findings will have a significant impact on basic cell and developmental biology and a deeper understanding of cell-cell communication and tissue development. In this way, we aim to control the spatiotemporal activation dynamics of Wnt signalling networks in vertebrate tissue. We envisage that in the longer term the results will, therefore, inform the development of novel tools to manipulate Wnt signalling pathways during development, wound healing and regenerative processes for the treatment of human disease.

Technical Summary

The Wnt signalling network is fundamental to development and tissue homeostasis in all multicellular organisms. Wnt proteins are produced in a source cell and act on the neighbouring cells to guide their behaviour. Despite three decades of Wnt research, it is currently unknown how these signalling proteins traffic between cells to activate the signalling pathway in the target cells. However, this knowledge is fundamental to control the Wnt network effectively during development, regeneration and disease. Our recent work has revealed the existence of an unexpected transport mechanism for Wnt proteins in vertebrates, which changes our understanding of how ligands spread in tissues. Wnts are rapidly loaded on thin cellular extensions called cytonemes to be transferred to target cells for signal activation. However, the mechanism of Wnt cytoneme trafficking - including the amount of Wnt protein transported - by cytonemes remains unknown. This project will provide new understanding of Wnt trafficking at the molecular level. Our hypothesis, supported by our preliminary data produced in preparation for this application, is that the number of Wnt proteins transported on a cytoneme and the number of receptors at the target cell controls the level of signal activation. We will test the hypothesis by (1) analysing the transport of Wnt ligands from producer to receiver cells by live cell imaging, (2) quantify Wnt components and their interactions at the cytoneme contact sites, and (3) develop a mathematical model to describe Wnt trafficking in vivo. The results of this multidisciplinary, multiscale project will provide a step change in understanding how the Wnt signalling pathway operates at the molecular and cellular level in a living vertebrate animal. This will open up new pathways to manipulate the Wnt signalling pathways during development, wound healing and regenerative processes for the treatment of human disease.

Planned Impact

This is a discovery science project, which aims to generate new quantitative knowledge regarding cell-cell communication in vertebrates. The work is timely because it will answer major unresolved questions in developmental biology and because the PI's team is developing ground-breaking analytical tools to study Wnt trafficking and signalling in an entire living organism. Currently, the PI has an international reputation for innovation in this field as the team has developed novel techniques to explore Wnt systems biology. The project will benefit the following beneficiaries and users with specific targeting and activities.

(1) Academic community: The results generated will be of immediate interest to scientists working in developmental and cell biology. The project will generate a set of mutant and transgenic zebrafish lines, and cell biological data, receptor-ligand measurements, and imaging data. All of the information will be made freely available, and lines will be archived both locally and at the European Zebrafish Resource Centre.

(2) Industrial partners: The results will be of interest to the microscopy industry in the field of super-resolution imaging. The PI has a strong track record of collaborative working with industry. The PI and Co-I have applied for a BBSRC CASE studentship with Leica as an industrial partner to develop fluorescent fluctuation techniques and has developed collaborative training programmes such as the EMBO microscopy workshop in 2013 supported by Leica and Bitplane. We further envisage significant impact on the pharmaceutical industry where there is a current focus on manipulating extracellular signalling to prevent metastatic cancer and other chronic diseases. This is of interest because it provides a completely new strategy for cancer treatment. The impact of research from this project will be realised through an active partnership with the relevant commercial sectors. At the start of the project, the PI will meet the University of Exeter IP & Commercialisation unit in the Innovation, Impact and Business Division to agree a strategy to protect and manage intellectual property and potential commercialisation opportunities that may emerge from the project. A plan will be agreed within the first six months of the project so that potential patent filing can be carried out ahead of publication, as detailed in the plan.

(3) Training for highly skilled researchers: Full training will be provided to the PDRA and the research technician in this project in specific skill development in cell and molecular biology, live cell imaging, and cell signalling, in addition to knowledge exchange and intellectual property management. The training includes close interaction with the University of Zurich to establish transgenic zebrafish lines and with the BU unit from Leica to further develop in vivo super-resolution microscopy. The PDRA will have the opportunity to present the work at national and international meetings.

(4) Wider public: Cell-cell communication and animal development are topics with a long history of captivating public interest and attention and have great potential to inspire the wider public about science and research. The concepts are readily understandable, easy to demonstrate and benefit from being highly visual. The PI has a wide range of experience dealing with media, i.e. contributions to radio, newspaper, and through the generation of YouTube videos. The PI is fully committed to developing the public understanding of science and will undertake communications through the popular press, as well as social media. (e.g. blog post in The Node, Oct. 2018) to a wide audience. Engagement activities with the University of the 3rd Age (U3A) will continue to take place twice per year as described. U3A engagement activities include lectures and hands-on investigative activities for retired and semi-retired people.

Publications

10 25 50
 
Description We have completed Objective 1a,b,c, and Objective 3b of the proposal and our manuscript has been accepted in Nature Communications.

Abstract: Wnt signalling regulates cell proliferation and cell differentiation as well as migration and polarity during development. However, it is still unclear how the Wnt ligand distribution is precisely controlled to fulfil these functions. Here, we show that the planar cell polarity protein Vangl2 regulates the distribution of Wnt by cytonemes. In zebrafish epiblast cells, mouse intestinal telocytes and human gastric cancer cells, Vangl2 activation generates extremely long cytonemes, which branch and deliver Wnt protein to multiple cells. The Vangl2-activated cytonemes increase Wnt/ß-catenin signalling in the surrounding cells. Concordantly, Vangl2 inhibition causes fewer and shorter cytonemes to be formed and reduces paracrine Wnt/ß-catenin signalling. A mathematical model simulating these Vangl2 functions on cytonemes in zebrafish gastrulation predicts a shift of the signalling gradient, altered tissue patterning, and a loss of tissue domain sharpness. We confirmed these predictions during anteroposterior patterning in the zebrafish neural plate. In summary, we demonstrate that Vangl2 is fundamental to paracrine Wnt/ß-catenin signalling by controlling cytoneme behaviour.

We have started to work on Objective 2a-c and 3a and have published a review article (Histochem Cell Biol. 2020 Nov;154(5):507-519).

Cell behaviour and function is determined through the interactions of a multitude of molecules working in concert. To observe these molecular dynamics, biophysical studies have been developed that track single interactions. Fluorescence correlation spectroscopy (FCS) is an optical biophysical technique that non-invasively resolves single molecules through recording the signal intensity at the femtolitre scale. However, recording the behaviour of these biomolecules using in vitro-based assays often fails to recapitulate the full range of variables in vivo that directly confer dynamics. Therefore, there has been an increasing interest in observing the state of these biomolecules within living organisms such as the zebrafish Danio rerio. In this review, we explore the advancements of FCS within the zebrafish and compare and contrast these findings to those found in vitro.
Exploitation Route The outcome of this proposal may shed light on the mechanism of how Wnt protein signals are distributed by cytonemes in vertebrate tissues - such as the developing zebrafish embryo.
Sectors Pharmaceuticals and Medical Biotechnology

 
Title Vangl2 promotes the formation of long cytonemes to enable distant Wnt/ß-catenin signalling 
Description Dataset 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact Not applicable yet. 
 
Description Cytoneme-based transport in gastric cancer 
Organisation Cardiff University
Country United Kingdom 
Sector Academic/University 
PI Contribution We have now investigated how cytonemes form using a combination of state-of-the-art genetic and high-resolution imaging techniques. We found that the Wnt proteins kick start their own transport; before they travel to their destination, they act on the cells that made them. A Wnt protein activates the receptor Ror2 and Vangl2 on the surface of the signal-producing cell. Ror2/Vangl2 then triggers signals inside the cell that begin the assembly of the cytonemes. The more Ror2 is activated, the more cytonemes the cell makes, and the more Wnt signals it can send out.
Collaborator Contribution Together with Prof Trevor Dale and Dr Toby Phesse, his mechanism operates in various tissues: Ror2/Vangl2 also controls the cytoneme transport process in living zebrafish embryos and human stomach tumours. This knowledge will help us to develop new ways to control Wnt signalling, which could help to produce new treatments for diseases ranging from cancers (for example in the stomach and bowel) to degenerative diseases such as Alzheimer's disease.
Impact Not yet.
Start Year 2018
 
Description Function of cytonemes in the mouse intestinal crypt 
Organisation Duke-NUS Graduate Medical School
Country Singapore 
Sector Academic/University 
PI Contribution We have now investigated how cytonemes form using a combination of state-of-the-art genetic and high-resolution imaging techniques. In initial experiments involving zebrafish cells that were grown in the laboratory, we found that the Wnt proteins kick start their own transport; before they travel to their destination, they act on the cells that made them. Wnt proteins activate the receptor Ror2 and Vangl2 on the surface of the signal-producing cell. Ror2/Vangl2 then triggers signals inside the cell that begin the assembly of the cytonemes. The more Vangl2/Ror2 is activated, the more cytonemes the cell makes, and the more Wnt signals it can send out. eLife : https://elifesciences.org/articles/36953 Nature Communications: accepted
Collaborator Contribution Together with the group of Prof DM Virshup, we have shown that cytonemes are regulated by Ror2 and Vangl2.This mechanism operates in various tissues: Ror2/Vangl2 also controls the cytoneme transport process in living zebrafish embryos, and in the mouse intestine. This knowledge will help us to develop new ways to control Wnt signalling, which could help to produce new treatments for diseases ranging from cancers (for example in the stomach and bowel) to degenerative diseases such as Alzheimer's disease.
Impact Publication in eLife, and Nature Communications. Collaboration is multi-disciplinary : cell biology, developmental biology, biochemistry.
Start Year 2017