Determining the functional order of Wnt signalling components

During development, groups of cells communicate with each other using protein messengers that are secreted from one cell and bind to receptors on neighbouring cells. One important family of protein messengers are called Wnts (pronounced wints). When the message is received, the responding cell switches on a number of genes in its nucleus that in turn bring about changes to processes such as growth and cell type. The connections inside the cell that transmit the Wnt signal from the cell surface to the nucleus are very important since they control how the signal is amplified and aimed at the right genes. The proposed research will use a method to the order in which connecting Wnt signalling components function inside the cell. The basic idea is to use a very sensitive cells that have been engineered to send out a burst of light when the Wnt signal is active in the nucleus. We then use a new method called RNAi to remove proteins that might be Wnt connecting components. If the protein IS required, the burst of light will be lost. This question is asked again and again for many genes using robots to speed up the process. We can ask whether and how many genes are required for Wnt signalling Once we know which proteins are involved in the Wnt pathway, we can study how they work and how they control animal development.

Wnt signalling controls a range of developmental processes including patterning, differentiation and cell proliferation. In cells responding to Wnt ligands, target gene transcription is regulated by a complex containing beta-catenin and the DNA binding factor TCF. Previous studies have identified up to 250 genes that may be involved in Wnt signalling in different systems. We propose to identify components of the Wnt signalling pathway in human 293 cells using an RNAi based screen. RNAi is a sequence-specific method of reducing target protein levels that has recently been developed for use in mammalian cells. RNAi screening studies of other signalling pathways have identified many novel components. We have optimised screening techniques in a highly sensitive TCF-reporter cell line and can immediately carry out a 96-well format screen of a 180 gene subset of putative Wnt signalling components. In collaboration with GE Biosciences (formerly Amersham; Cardiff), we will establish further Wnt signalling assays using a new machine capable of high throughput confocal laser microscopy. The combination of these cutting edge techniques will be used to establish the functional order of Wnt signalling components by genetic epistasis. At a technical level, this work should establish a platform of expertise for further high throughput RNAi/Cell line based screens in the UK. At the scientific level, this work will order novel and existing Wnt components with respect to each other and should identify new branches to the existing molecular Wnt pathways. Results from the screens (including microscopic images of RNAi treated cells) will be stored in a form that is compatible with gene-specific databases and will be accessible to the scientific community through a web-accessible relational database.