Molecular regulation of thymic epithelial lineage specification

Abstract

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.