The regulation of early thymocyte development by morphogen signalling

T cells are white blood cells that are made in an organ called the thymus. T cells are essential to immunity because they both fight infectious diseases and instruct other types of white blood cell to fight infection. Understanding how T-cells are produced is therefore very important to our understanding of immunity. When developing T-cells divide out-of-control T cell leukaemias and lymphomas arise, so understanding what controls their production will also help us to understand and design treatments for leukaemia. This project will study how molecules called 'Hedgehog' proteins control the production of T cells in the thymus. Hedgehog proteins are molecules that are made by cells and secreted to tell other neighbouring cells to undergo a particular process, such as to divide, die or change into a different type of cell. They do this by controlling which molecules are made in the target cell, and this is determined by which genes are active in that cell. Hedgehog proteins are essential in our embryonic development because they transmit messages to developing organs controlling how the organs form and grow. We have shown that after birth Hedgehog proteins are also important in our immune systems because they allow communication between different cells of the immune system. They also determine how many T-cells are produced in the thymus and they can make developing T-cells divide, or stop dividing. We aim to find out what messages Hedgehog proteins give to developing T-cells in the thymus by finding out which molecules are made and which genes become active when Hedgehog proteins signal to T-cells at different stages of their development.

This project will investigate the regulation of early T-cell development in the thymus by Sonic hedgehog (Shh) and Indian hedgehog (Ihh). Shh and Ihh signal for proliferation, survival and differentiation of early CD4-CD8- double negative (DN) thymocyte progenitors, but negatively regulated pre-T cell receptor (TCR)-dependent differentiation from DN cell to CD4+CD8+ double positive (DP) cell. This project will investigate the molecular mechanisms and Hh-target genes that underlie the positive regulation and later negative regulation (after pre-TCR signal transduction) of thymocyte development by Hh signalling. Our general experimental approach will be to probe Ihh and Shh function in mouse mutants in which Hh protein production and secretion by thymic epithelium and thymocytes, or the ability to respond to the Hh signal in thymocytes, are specifically ablated/inhibited. We will use these mouse models in conjunction with Rag1-/- mice, in which pre-TCR signal transduction can mimicked by treatment with anti-CD3 antibody, to investigate the impact of Hh signalling on DN thymocyte differentiation, proliferation and survival in the absence of pre-TCR signalling, and on pre-TCR-induced differentiation to DP cell. We will test the hypothesis that Ihh produced by DP thymocytes feeds back to negatively regulate the differentiation of their DN precursors. We will investigate the interactions between pre-TCR signal transduction and Hh pathway activation in developing thymocytes. We will identify and compare target genes of the Hh signalling pathway in developing thymocytes at the earliest stages of thymocyte development, when Hh signalling promotes T-cell development, with those at the transition to DP cell, when Hh pathway activation is a negative regulator of thymocyte differentiation. We will test the hypothesis that the transcription factors FoxA1 and FoxA2 are negative regulators of differentiation to DP cell, downstream of Hh signalling.

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 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. In addition, our collaborator, Professor Georg Hollaender, is also a Consultant Paediatric Immunologist, who works at The University Children's Hospital of Basel, Switzerland and so provides another opportunity for us to disseminate our findings to influence human health. (2) Industrial Partners Past findings from our studies on Hedgehog signalling in T-cell development were patented world-wide (1999, 2004) 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, and we also benefit directly from UCL's Intellectual Property expertise. (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 familiarise 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 Susan Outram, and by other lab members. Collaboration We will collaborate with Prof Georg Hollaender, The University Children's Hospital of Basel, Switzerland. Georg Hollaender runs a world-leading laboratory working on thymic epithelium. For this project he will supply us with Fox-N1-Cre transgenic mice to allow excision of 'floxed' alleles (Disp1, Shh, Ihh) from mouse thymic epithelial cells. Georg Hollaender will also provide reagents and technical guidance for the analysis of thymic architecture and thymic epithelium in the conditional Dips1 models. The Hollaender lab has carried out extensive gene expression studies in mouse thymic epithelial cell populations, and this data will inform our project, particularly for our analysis of thymic architecture in the conditional Disp1 models.