The function of the FoxA transcription factors in T-cell development and homeostasis

Background:
The thymus provides a specialized environment that supports the maturation of lymphocyte precursors into functional T-cells. The thymus is seeded by blood-borne progenitor cells from the foetal liver or adult bone-marrow which develop along a wellcharacterised programme, dependent on bidirectional signals between the developing thymocytes and the thymic epithelial cells (TEC).

We identified the transcription factors FoxA1 and FoxA2 to be downstream of Hh pathway activation (Hh target genes) in thymocytes and peripheral T-cells and we have recently shown that FoxA1 and FoxA2 are also important for TEC development and function1-3. These transcription factors are essential for mouse development and mutation leads to severe defects in node, notochord, neural tube and gut4. We have therefore obtained floxed FoxA2 and FoxA1 mice in order to generate conditional FoxA2-deficient animals in which FoxA2 is excised form T-lineage cells at different stages of T-cell development.

Aims/Objectives:
The purpose of this project is to test the hypothesis that the transcription factors FoxA2 and FoxA1 are required at the transition from CD4-CD8- double negative (DN) cell to CD4+CD8+ double positive (DP) cell, for efficient production of CD4 single positive (SP) cells in the thymus, and for T-cell homeostasis and function in the periphery. We will complete the following objectives:
1. We will test if conditional deletion of FoxA2and/or FoxA1 from T-lineage cells influences differentiation from DN to DP cell, and from DP to CD4SP and positive selection.
2. We will identify FoxA2/A1-target genes in T-lineage cells at different stages of their differentiation.
3. We will test if FoxA2/A1 is required for homeostasis and function of peripheral T-cells.

Methods:
This project will use conditional mutant mouse models, in combination with in vitro functional assays and immunisations to investigate FoxA function in immunity. The project will involve flow cytometry, microscopy, molecular biology, RNA-sequencing and bioinformatics, computational analyses, using methods described in 1-3,5.

Timeline:
Year 1: analysis of thymocyte development in FoxA1/A2 deficient and double deficient mice;
Year 2: studies on peripheral T-cell function and homeostasis;
Year 3: Identification of target genes; preliminary investigation of function of target genes.

References:
1. Lau CI, Yánez DC, Solanki A, Papaioannou E, Saldaña JI, Crompton T. (2018) Foxa1 and Foxa2 in thymic epithelial cells (TEC) regulate medullary TEC and regulatory T-cell maturation. J Autoimmunity 93:131-138.
2. Solanki A, Lau C-I, Saldana JI, Ross S and Crompton T. (2017) The transcription factor Gli3 promotes B cell development in the fetal liver through repression of Shh. Journal of Experimental Medicine 214, 2041.
3. Rowbotham NJ, Hager-Theodorides AL, Furmanski AL, Ross SE, Outram SV, Dessens JT and Crompton T. (2009) Sonic hedgehog negatively regulates pre-TCR induced differentiation by a Gli2 dependent mechanism. Blood 113, 5144.
4. Ang SL, Rossant J. HNF-3 beta is essential for node and notochord formation in mouse development. Cell. 1994;78:561-574.
5. Solanki A, Lau C-I, Saldana JI, Ross S and Crompton T. (2017) The transcription factor Gli3 promotes B cell development in the fetal liver through repression of Shh. Journal of Experimental Medicine 214, 2041.