The mechanism of GPCR signalling in zebrafish semicircular canal morphogenesis

The semicircular canals of the inner ear detect turning movements of our head, triggering muscular reflexes that enable us to maintain our balance. There are three canals in each inner ear, and these are each arranged at right angles to one another, so that they can detect movement in any direction. Understanding how these canals develop in the embryo, together with the rest of the intricate structure of the inner ear-rightly also known as the labyrinth-is an interesting problem. We study this process in the zebrafish embryo, but there are many similarities to the sequence of events during human development. The zebrafish ear initially starts out in the early embryo as a simple fluid-filled ball of cells. Projections of tissue then form that grow out into the fluid-filled space inside this ball, where they touch and fuse to make pillars, which become the hubs of the semicircular canal ducts. We have recently identified a receptor protein that appears to be involved in triggering a whole series of events to enable to the projections to recognise one another, fuse and rearrange accordingly. When the gene coding for this receptor is disrupted, the projections fail to touch and fuse: they appear to slide past one another without recognition. As a result, the semicircular canals do not form properly, the ear becomes misshapen and swollen, and the fish grow up with mild balance defects. The receptor is a member of an interesting class of proteins called GPCRs that are located in the cell membrane, projecting from its surface. A GPCR receptor does not act in isolation, however, but usually responds to a signalling molecule at the cell surface, and then triggers events inside the cell that activate new genes. One of the aims of this project is to identify the other players in this process. We have a number of good clues from fish strains in which the semicircular canals fail to form correctly. We will aim to identify any new genes involved in the same process. We will also gather information about when and where the receptor protein is present, how it is regulated, and how it functions during the development process. We have already identified some of the genes that are regulated by the receptor. One class of these, the versicans, remain highly active when receptor function is missing, when they should normally be turned off. We will test whether the versicans are the players that actually control how the projections move and fuse in the developing zebrafish ear. A final aim is to search for small drug-like molecules that can affect the process of semicircular canal formation in the ear. There are many GPCR proteins, and these comprise targets for many of the drugs that are on the market today. There is, therefore, considerable commercial interest in understanding more about GPCRs and in identifying new drugs that affect their activity. We will aim to carry out a small-scale study to identify new activators and inhibitors of the receptor, which we will then be able to develop further in future projects. Although this project aims to understand processes that happen in the zebrafish ear, the GPCR is active in other areas of the embryo, and so the conclusions are likely to aid in our understanding of the development of other organ systems, such as the heart and nervous system.

Morphogenesis of the semicircular canal ducts in the vertebrate inner ear is a dramatic example of epithelial remodelling in the embryo. We have recently identified a completely new receptor involved in semicircular canal formation in the zebrafish ear. The gene is gpr126, an adhesion class G protein-coupled receptor gene. In lauscher (lau) mutant zebrafish embryos, which lack gpr126 function, epithelial projections grow out in the ear, but fail to fuse with one another to form pillars. A variety of extracellular matrix components, including versican genes, are hugely upregulated in the unfused projections, and the ear becomes grossly swollen. This proposal uses multidisciplinary approaches to dissect the role of the Gpr126 pathway in semicircular canal formation in detail, and set it in a wider context. Specifically, we aim to identify new pathway members through the cloning of strong candidate mutants and genes. We will characterise the Gpr126 protein and explore its mode of action through transplantation experiments, and test interaction of the Gpr126 pathway with Hh signalling in the ear. We will test the hypothesis that the versican gene products play an anti-adhesive role in the process of projection outgrowth and pillar formation in the zebrafish ear. Lastly, we will perform a pilot chemical screen, using versican expression as an assay, to identify novel agonists and antagonists of the pathway. The proposed research is timely. It exploits the findings described in our submitted manuscript on the otic phenotype of the lau mutant, and links with our published BBSRC-funded work on Hh signalling in the ear. The work will characterise a newly identified mutant provided by our collaborator, Dr J. Topczewski, and will also exploit a new unpublished transgenic line, provided by Dr R. Knight. The project also utilises our new MRC CDBG screening facility, which has been established with MRC Pump Priming funds to support projects such as this one.

Who will benefit from this research? In adding to the scientific knowledge base, our work will benefit a wide range of academics. These include the PDRA, other members of the Whitfield research group, our collaborators, and other developmental biologists working on the ear and other aspects of epithelial morphogenesis. The work will also be of benefit to pharmacologists, protein biochemists and structural biologists working on GPCR signalling. Further detail is given in the 'Academic Beneficiaries' and 'Pathways to Impact' section. Beneficiaries in the commercial private sector include companies working on GPCRs as a route to drug discovery and design. Further detail is given in the 'Pathways to Impact' section. The MRC CDBG and Department of Biomedical Science have an active programme of outreach activities. We will aim to maintain an active profile in outreach work, in order to benefit the wider public. In the past, I have enjoyed presenting our work to the public at exhibitions, school visits and departmental open days. Members of my lab have also made contributions to outreach events; Dr Hammond (Researcher Co-investigator on our current BBSRC grant) has been particularly active in this area. I will encourage new members of staff to play a similarly active role in our outreach activities. How will they benefit? We will inform other academics of our work through normal channels (publications and presentations at meetings and seminars). Colleagues will benefit from an increased understanding of the developmental processes underlying semicircular canal formation and the role that the Gpr126 pathway and versicans play in this process. We will work with existing collaborators and establish new collaborations for the development of further projects and proposals. This will be done throughout the project as opportunities arise, depending on the data generated. Compounds identified through our screening programme, and our automated assays for screening, will be of benefit to companies developing therapeutic products based around GPCR signalling. Dr Smith will provide advice on IP protection and if necessary we will put confidential disclosure agreements in place. This will be done when results from the screening programme (Objective 4) are realised. The public will benefit from an increased knowledge and understanding of scientific research and its application, and the opportunity to meet and talk with research scientists. Examples of our past activities include a presentation at the Royal Society Summer Science Exhibition (2009), with a variety of engaging and fun activities based around research with zebrafish embryos. We hope that through activities like these we will encourage all young people to develop their interests in science, and attract some to pursue scientific careers. Skills The PRDA and technician will benefit from training in a wide variety of lab techniques, including molecular biology, compound and confocal microscopy, chemical screening assays, histological techniques and embryological manipulations. At the end of the project, they will be well equipped to seek further employment in bioscience labs in either the academic or commercial sectors. As examples, former students from the Whitfield lab have taken up postdoc positions in the US, and a technician has recently moved on to take up employment in a private fertility clinic.