The genetic mechanisms underlying the specification of thymic epithelial (TE) lineage cells are poorly understood. Foxn1 is an early specific marker of thymic epithelial cells (TECs) in the third pharyngeal pouch (3PP) and is required for development of all mature TE lineage cells but does not specify the TE lineage. The upstream regulators of Foxn1 are currently unknown, however evidence points to a potential role for Pax1 and Pax9. While the thymus phenotypes of the Pax1-/- and Pax9-/- mutant mice have been investigated in detail and TECs in these mice are known to express Foxn1, the possibility of functional redundancy exists and the compound mutants of these genes have not been studied. Therefore, the aim of this thesis was to test the hypothesis that Pax1 and Pax9 are required for TE lineage specification and regulation of Foxn1 expression. This hypothesis was addressed by analysis of thymus development and TEC function in Pax1/Pax9 compound mutant mice. The data presented in this thesis indicates that prenatally, Pax1 and Pax9 cooperatively regulate thymus organogenesis, such that the size, structure and location of the thymus is affected in a Pax1/Pax9 gene dosage-dependent manner, and the Pax1unex/unexPax9lacZ/lacZ embryo is functionally athymic. Furthermore, they establish that the thymic rudiment of Pax1unex/unexPax9lacZ/lacZ embryos does not express Foxn1, establishing that Pax1 and Pax9 are required together for the initiation of Foxn1 and suggesting they are required to specify the TEC lineage. Postnatally, enlarged blood vessels observed in the Pax1unex/unex thymus suggested a role for Pax1 in vascularisation of the thymus. In addition, the effect of loss of one or more Pax1/Pax9 alleles on the expression of Foxn1 and other genes known to regulate TEC development or function was assessed. These data demonstrate that Pax1 and Pax9 co-operate to regulate Foxn1 in a dosage-dependent manner. Furthermore, Pax1 and Pax9 appear to negatively regulate both Hoxa3 and Vegfa, providing a possible explanation for the enlarged blood vessels in the postnatal Pax1unex/unex thymus. Finally, an inducible and reversible recombinase-mediated cassette exchange system that will allow the knockdown of Pax1 and Pax9 at defined time points during development has been established, that has the potential to test the function of these genes during thymus organogenesis and in the postnatal thymus.

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Central to the age-associated decrease in immune system function, characterised by the increase in the frequency and severity of infections and autoimmune diseases, is the decrease in production of naïve T cells by the thymus. This results from the targeted degeneration or involution of the thymus with age. One of the principal causes of involution is the loss of organisation and functionality of the thymic epithelium, which confers the primary function of the organ via interactive regulation of T cell development. Although the mechanisms that govern the deterioration of the thymic epithelium are poorly understood, a number of recent reports indicate that the transcription factor, Foxn1, is required to maintain this compartment in the postnatal thymus. Thus, the first aim of this study was to precisely profile Foxn1 expression levels in aging postnatal thymic epithelial cells. The second aim was to investigate the effects of upregulating Foxn1 in the aging thymus, which was achieved using a novel, regulatable Foxn1 mouse model generated during this study. In this study I show that Foxn1 is expressed at different levels in different postnatal thymic epithelial cell (TEC) sub-populations suggesting a dosage-dependent mode of action for Foxn1. Additionally, using two experimental approaches, I show that Foxn1 expression decreases with age in TECs, supporting the current data that implicate the loss of Foxn1 as a potential cause of thymus involution. Next, I generated a tissue-specific, regulatable Foxn1 mouse model that allowed me to modulate Foxn1 expression in the postnatal thymus. Firstly, using this model, I show that thymus involution can be prevented by the up-regulation and maintenance of Foxn1 expression from the onset of involution. Thymi that up-regulated Foxn1 were overtly larger and exhibited greater cellularity in both the thymocyte and epithelial compartments compared to age matched controls. Additionally, the larger TEC compartment contained a higher proportion of functional and proliferating TECs that upregulated a panel of genes involved in TEC development and function. Next, I show that Foxn1 up-regulation in aged, involuted thymi is sufficient to partially reverse involution, as shown by an increase in TEC organisation and intrathymic T cell numbers. While other strategies that promote thymic rebound or reversal have been reported, including cytokine treatment or sex steroid ablation, these approaches are complicated by side effects and toxicity. Hence, I propose a novel model for immune reconstitution through the regulation of Foxn1 expression in the postnatal thymus.

No / Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.