The Gli family of transcription factors in thymic epithelial cell (TEC) function and differentiation

T-cells are white blood cells that enable us to fight infectious disease. To do this, T cells must be able to recognize what is 'self' from what is 'non-self'. T-cells are produced in an organ called the thymus. During their maturation in the thymus, T-cells are selected so that any T-cells that might attack our-selves (attack 'self') die, but T-cells that are able to fight infections are allowed to leave. This project will study how the thymus controls which developing T-cells are allowed to complete their maturation and leave the thymus because they recognize 'non-self' and which T-cells do not complete their maturation because they recognize 'self'. We will study how three molecules called Gli1, Gli2 and Gli3 control the fate of developing T-cells and influences their ability to recognize 'self' and 'non-self'. Gli1, Gli2 and Gli3 are molecules called a transcription factors that influence which genes are active in a cell. We will find out which genes are controlled by Gli1, Gli2 and Gli3 in the thymus. We will use this information to find out how Gli1, Gli2 and Gli3 control the production of T-cells.

This project will investigate the functions of the Gli family of transcription factors, Gli1, Gli2 and Gli3, in thymic epithelial cells (TEC) and the thymus stroma. We will investigate how the Gli proteins influences TEC differentiation and function, and how this impacts on T-cell development and the induction of tolerance. We will test the hypothesis that it is discrete subsets of TEC that express Gli1, Gli2 and Gli3. We will define these subsets, and investigate the part played by Gli proteins in their development and function. In parallel, we will investigate the way in which the Gli transcription factor's functions in TEC influence T-cell development, and in particular their influence on T-cell receptor (TCR) repertoire selection and differentiation from CD4+CD8+ double positive thymocyte to mature CD4+ and CD8+ single positive (SP) cell. Our general experimental approach will be to probe Gli function in mouse mutants in which Gli2 or Gli3 is specifically deleted in TEC, and in radiation bone-marrow chimeras and reaggreagate thymic organ cultures in which the stromal compartment of the thymus is Gli1-deficient and the haematopoietic compartment wild type. We will analyse TEC differentiation and function, thymus architecture and TEC organisation. We will use microarray analysis of transcription in TEC populations from these models to identify Gli1-, Gli2- and Gli3-target genes and Shh target genes in TEC. We will model the transcriptional response of TEC to Shh in the presence or absence of Gli3. We will investigate the functions of these novel Gli and Shh targets in TEC. We will test the impact of Gli-deficiency from TEC on T-cell development and tolerance induction in these mice. We will use the Gli-deficient mouse models in conjunction with TCR-transgenic mice to investigate the impact of Gli expression by TEC on TCR repertoire selection and CD4/CD8 lineage commitment in thymocytes, and tolerance induction.

Our work can impact on human and animal health, and we will therefore communicate and engage with the Health services and with the Pharmaceutical/Biotechnological industry. In addition, we are actively involved in Public Engagement in Science. Communications and Engagement (1) The Health Services We are well positioned to engage with the Health services and to disseminate our findings to promote up take by the Health services because ICH is situated at Great Ormond Street Hospital (GOSH). We frequently attend and present at ICH/GOSH seminars and meetings, where we discuss our data directly with clinicians working within the Health service at GOSH. For example, we collaborate with Dr Graham Davies (Consultant Paediatric Immunologist, GOSH) who is currently establishing thymus epithelium transplant for the treatment of DiGeorge syndrome in the UK. Our research in mouse models will directly inform his research into the culture and preparation of human thymic epithelium for transplantation. (2) Industrial Partners Past findings from our studies on Hedgehog signalling in the thymus were patented world-wide and commercially exploited by the American Biotechnology company, Curis. Assignment of the patents were sold to Curis by Imperial College London. We have maintained good informal links with Curis, who will provide us with reagents if necessary, and we continue to discuss ideas with Curis scientists. In the future, Curis may again purchase licence of patents from our work. Our contracts and negotiations with industrial partners will be handled by ICH/GOSH Research and Development (R&D) Office. The R&D Office will advise on Intellectual Property rights and forge links with industrial partners. (3) Public Understanding of Science In the interests of public engagement in science, my group has established links with local London primary and secondary schools. We organise day workshops at primary schools and also day workshops in our laboratory for secondary school students. These workshops allow us to explain our work to the students in lay terms, and we also introduce the students with an experimental approach to hypothesis testing in science, and in the case of secondary school students, to some of the experimental techniques of our work, including flow cytometry, PCR and microscopy. We will continue with these activities and present this project to the students in lay terms. We also provide work experience for older secondary school students, giving them a chance to 'shadow' a postdoctoral worker for a day or for a week, and we will continue with this activity. These activities will be carried out by both Tessa Crompton and Anna Furmanski and by other lab members. Dr Susan Ross (a postdoctoral scientist in the group who also has a PGCE teaching qualification), is currently a Wellcome Trust scientist in residence at the Wren Academy in North London, where she is arranging workshops on Immunobiology. Collaboration We will collaborate with Dr Mike Hubank and Dr Martino Barenco, from ICH. Martino Barenco is an MRC Methodology Research Fellow, with a background as a mathematician. Martino Barenco and Mike Hubank have developed mathematical methodology that enables the analysis of microarray technology following RNA accumulation and degradation through time following a stimulus, in order to identify profiles of transcriptional activity which account for the functional consequences of that stimulus. They will collaborate with us to apply this systems approach to model the transcriptional response of thymic epithelial cells to Sonic hedgehog in the presence or absence of Gli3. Dr Hubank is director of UCL genomics and he will also collaborate with us on carrying out the microarray experiments (Quality control, microarray platforms, bioinformatics). Prof Georg Hollaender, Oxford, will supply us with Fox-N1-Cre transgenic mice to allow excision of 'floxed' alleles from thymic epithelial cells.