Investigation of a completely uncharacterised, embryonically lethal gene in immune cell development

The development of vertebrate animals from a single fertilised cell to the fully formed adult is an astonishingly well-choreographed sequence of events, which is made more remarkable by being so faithfully recapitulated on each attempt. One part of this sequence is the development of an immune system, which is essential for the animal to remain healthy whilst being continuously exposed to pathogens throughout its life. It follows though that when these developmental processes do go wrong, there can be serious negative impacts on survival.

T cells are an essential white blood cell-type of the immune systems of many animals, including our own. They are responsible for both the detection and elimination of many pathogens, including bacteria, viruses and cancerous cells. The specificity and function of T cells are instructed during their development in the thymus. During this time, the immature T cells transiently express the pre-T cell receptor (pre-TCR), which orchestrates the first stage of T-cell development. Elucidating pre-TCR function is necessary both for a fundamental understanding of immune cell biology, but also for how we might be able to manipulate its signalling therapeutically when it goes wrong, as is the case in some leukaemias. However, studying the molecular details of the pre-TCR is hindered by the temporary nature of its expression and that these immature T cells are extremely difficult to remove from living tissue. To overcome this challenge, my group has recently reconstituted expression of the complete pre-TCR complex in non-immune cells, so that its fundamental biology can be explored in a far more tractable manner. We have replicated known features of the pre-TCR, principally its constitutive internalisation from the cell surface.

Through this work, we have identified a completely uncharacterised protein (‘UP1’) that is important in this process. Unexpectedly, we have found that knocking out the gene encoding UP1 in the mouse causes a complete loss of viable offspring, showing that its function must be essential more generally in embryonic development. The only available evidence for ascribing a function to UP1 comes from genetic association studies, where it has been found to be upregulated during thymic development, to have dysregulated methylation in patients with chromosome-21 trisomy (Down’s Syndrome) and its upregulation has been implicated in the development of neurological disorders such as dementia and Alzheimer’s disease. These results suggest a possible role for UP1 in the functioning of neurons as well as in the immune system. Pertinently, the UP1 gene is present and extremely well conserved throughout the vertebrate lineage. For example, the zebrafish homologue shares 72% identity, 84% similarity at the protein level. High sequence conservation through evolution is normally strong evidence that the protein has an important role to play in the function of the organism.

The central goal of this project is to begin uncovering the molecular functions of UP1, with the hypothesis that it is a chaperone that helps other developmentally important protein complexes to form correctly. We will characterise the molecular details of UP1 in a reconstituted cellular system and in the zebrafish model organism where embryo development can be directly observed. It is unusual to identify a completely uncharacterised protein with no known structure or function, which is nonetheless essential for development. I believe these characteristics, along with the opportunities for extending the research in myriad different directions, provide a very exciting new research project for a PhD student.
Techniques that will be undertaken during the project:
- Molecular biology and cloning
- Cell Culture
- Flow cytometry
- Spinning-disk confocal microscopy
- Quantitative Western analysis
- Zebrafish developmental biology