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A Foxn1 reporter knock-in mouse for thymic epithelial research
thesisposted on 2017-02-09, 02:40 authored by Lim, Joanna Mei Ch'wan
The thymus is critical for the development of mainstream T cell mediated adaptive immunity. A key stromal component is the thymic epithelium, which can be broadly divided into cortical and medullary thymic epithelial cells (cTEC and mTEC). Developing thymocytes migrate through and interact with specialised TEC subsets in a co dependent manner, known as cross talk. Despite its immunological importance, the thymus paradoxically involutes with age and is proposed to contribute to the clinical symptoms of immunosenescence. Thymic epithelium, is unique as it begins to atrophy soon after birth but, under appropriate stimuli, is able to regenerate partially following damage, at least in the young to middle aged. This probably involves resident thymic epithelial progenitor cells (TEPC), but to date they have not been identified in the adult. Investigations into thymic development, maintenance in the adult and the capacity to regenerate are critical for developing clinically relevant strategies to treat immune degenerative conditions. Such studies will also serve as important proof of concept contributions to regenerative medicine in general. The Forkhead box transcription factor n1 (Foxn1) is the best characterised and earliest known TEC regulator, marking bipotent TEPC in the embryo and required for the maintenance of TEC and their sub lineages. In this project, we generated Foxn1eGFP/w knock in reporter mouse embryonic stem cell (mESC) lines in two strains (C57BL/6 and 129/Sv) and a corresponding C57BL/6 mouse line, to assist in the study of TEC development and maintenance at all life stages, and the role of Foxn1 in these processes. The Foxn1eGFP/w mouse described in this thesis is currently the only fluorescent reporter knock-in mouse model for Foxn1. In the Foxn1eGFP allele, eGFP is under the transcriptional control of the endogenous Foxn1 promoter and regulatory elements, ensuring accurate reporter expression compared to other transgenic mouse models. Its main advantage over existing Foxn1LacZ/+ reporter mice is the ability to identify, isolate and characterise viable eGFP+ cells, which are exclusively in epithelial cells of the thymus and skin. The generated young Foxn1eGFP/w heterozygotes were phenotypically normal and indistinguishable from Foxn1w/w mice with normal fur development, thymic cellularity, TEC and T cell proportions and numbers. Foxn1eGFP/eGFP mice phenocopied conventional BALB/c Foxn1nu/nu (nude) mice, with thymic dysgenesis, hairlessness and T cell defects. eGFP expression reflected reported Foxn1 expression throughout ontogeny. There was a very close correlation between Foxn1 and eGFP at the protein and mRNA levels. The majority of TEC in the young and middle aged Foxn1eGFP/w mice are eGFP+ confirming previous reports that the majority of TEC arise from a Foxn1+ lineage. eGFP- TEC in the adult could also represent immature TEC that express eGFP and Foxn1 mRNA and are still in the process of translating eGFP and Foxn1 protein or they could be apoptosing TEC. Early studies in our Foxn1eGFP/w mice show a possible reduction in Foxn1 and eGFP mRNA in aged but not middle aged TEC, compared to young levels. Collectively, the Foxn1eGFP/w colony reflects the reported Foxn1LacZ/+ colony with the added benefit of isolating and culturing viable eGFP+ TEC in vitro. Mild haploinsufficiency as previously documented in Foxn1nu/+ heterozygotes, was not observed in young Foxn1eGFP/w steady state thymii. Furthermore, Foxn1eGFP/w thymii, despite having reduced Foxn1 levels, responded in a similar manner to wildtype thymii in preliminary studies of damage induced thymic regeneration and castration induced thymic hypertrophy. The Foxn1eGFP/w reporter mouse colony is a useful tool in thymic epithelial research at all life stages - from the technical isolation of early embryonic or Foxn1eGFP/eGFP “nude” thymii, characterising adult and aged TEPC, establishing in vitro TEC cultures and determining the precise roles for Foxn1 in TEPC and TEC maintenance and ageing.