Role of foxn1 in Xenopus laevis thymopoiesis.

Abstract

Abstract The transcription factor forkhead box N1 (FOXN1) plays a pivotal role in differentiation of thymic epithelial cells (TECs) essential for intrathymic T cell education and keratinocyte development. Mutations in murine foxn1 are associated with T− B+ NK+ nude/severe combined immunodeficiency (SCID) phenotype. In humans FOXN1 mutations also lead to thymic aplasia or hypoplasia, hair loss and dysmorphic nails. In the amphibian Xenopus tropicalis, foxn1−/− line exhibited loss of thymi formation and absence of mature T-cells, similar with Foxn1−/− murine line. Due to expanded resources in immunobiology of X. laevis (a close relative of X. tropicalis), we generated a novel foxn1-deficient X. laevis model by using the CRISPR/Cas9. foxn1 crispants shows a high mutational efficiency (30–90%) and about 25% of tadpoles (stage 55–56), exhibit hypoplastic thymi. foxn1 disruption resulted in significant reduction of T-cell markers transcript levels, detected by RT-PCR. Furthermore, spleen of foxn1 crispants had markedly reduced CD3e staining compared to controls. Interestingly, Mycobacterium marinum (M. marinum) infection of tadpoles results in noninflammatory CD8 negative- and iT cell–mediated response, while foxn1 mutations in X. laevis appeared to affect tolerigenic innate immune response of infected tadpoles. This foxn1-deficient transgenic line will empower X. laevis as nonmammalian alternative experimental organism for studying host interaction with to non-tuberculosis mycobacteria. Currently, we are investigating mechanisms by which foxn1 mutation alter innate immunity of M. marinum inoculated tadpoles and evaluating tolerance/resistance by analysing the inflammatory/anti-inflammatory cytokine profile.