PAR-1, polarity and Wnt signalling in early vertebrate embryos

There are good reasons to hope that in the future it will be possible to regenerate damaged or diseased parts of the body using the same natural chemicals that generate the structure of the body during normal development of the embryo. For this to be a possibility, it is first essential to get a detailed understanding of how those chemicals, known as morphogens, do their job. This project proposes to analyse a particular morphogen known as Wnt. Wnt is used, among other things, (a) to trigger the switching on of particular genes that make the head and front end of the body and (b) to direct the movements of cells within the embryo so that it is long and narrow (rather than short and round). These two processes happen quite quickly after one another and it is not properly understood how the many cells involved in both processes are able to respond differently to the same Wnt signal. We have been studying another protein, PAR-1, that may help us understand the different Wnt responses. PAR-1 is a protein that exists within cells that makes one end of a cell different from the other. A layer of such cells (known as ?epithelia? e.g. the lining of your gut or kidney) can therefore have one surface be very different from the other (i.e. have epithelial polarity). We believe that Wnt arriving on one surface may act differently from Wnt arriving at the other surface. PAR-1 may be important in this because different versions of the PAR-1 protein react differently to Wnt and may be localised to opposite surfaces of epithelia. To test this, we will (1) check to see if different versions of PAR-1 are indeed localised to the different surfaces, and if so which parts of the structure of PAR-1 enable this differential localisation; (2) determine whether the Wnt-induced cell movements mentioned above, which we know are affected by PAR-1 removal, are affected because the epithelial polarity is disrupted; and (3) find out what PAR-1, which is an enzyme that modifies other proteins, actually does (i.e. which other proteins it modifies), both by testing some candidate proteins and screening for other protein targets. These experiments will help work out the ?wiring diagram? for cell regulation in embryos and ultimately enable cells to be manipulated for therapeutic outcomes.

The Wnt signalling pathways are highly conserved and are of major significance in development and disease. Canonical Wnt signalling activates transcription of specific target genes, such as those that initiate the Spemann Organiser in amphibian embryos, while non-canonical Wnt-PCP (planar cell polarity) signals regulate the convergent extension (CE) movements of gastrulation that elongate the primary body axis and the developing inner ear. The mechanism of Wnt-PCP signalling in CE and the regulation of canonical versus non-canonical signalling remain important unsolved problems in the developmental biology and signal transduction fields. Using Xenopus embryos, we have shown that the polarity protein PAR-1 is a critical component of either the canonical or the non-canonical planar cell polarity (PCP) Wnt signalling pathways, depending on the particular PAR-1 isoform. Understanding isoform differences will therefore inform our understanding of the different Wnt pathway branches. PAR-1 is associated with the apicobasal polarity of epithelia and there is evidence in Drosophila that canonical Wnt and non-canonical PCP signalling may be separated by differential localisation of components in the apicobasal axis. However, this model has not been tested in a vertebrate system. We have preliminary data indicating that PAR-1 isoform differences differentially localise the proteins apicobasally in a manner consistent with this idea. We propose, therefore to test the structural basis of this differential localisation by deletion and fusion-chimera analysis of the different isoforms as a first step in testing this model. Secondly, to better understand the mechanism of action of PAR-1 in PCP signalling and CE, we will conduct a detailed analysis of the PAR-1-depletion phenotype, specifically the effects of such depletion on polarised fibronectin deposition, PCP-protein localisation and cellular protrusivity/orientation that represent three key phases of CE. Finally, we will identify relevant targets of PAR-1, since we have evidence that not only Dishevelled protein but also other components of the Wnt canonical and PCP pathways are targets for PAR-1 kinase activity. To this end, we shall test candidate target proteins (especially GSK3-beta) as well as conducting an unbiased in vitro screen to identify new targets in a normalised Xenopus expression library. Positive ?hits? will be validated both in vitro for phosphorylation and in vivo for a depletion phenotype in Xenopus embryos. Easy microinjection, well-validated target protein depletion and large cell size make Xenopus the most advantageous system for the high-resolution morphogenetic and molecular imaging in both the planar and apicobasal dimensions required for this project.