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

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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.