Characterization of Lgr5‐positive epithelial cells in the murine thymus

Abstract

Leucine-rich repeat-containing G protein-coupled receptor (Lgr)5 is a marker for epithelial stem cells in the adult intestine of mice. Lgr5 transcripts have also been detected in the developing murine thymus, leading to speculation that Lgr5 is a marker for the longsought stem cell of the thymus. To address the nature of the Lgr5-expressing thymic epithelial cells (TECs), we used Lgr5-GFP reporter mice. We show that epithelial cells expressing Lgr5 protein are present in the fetal thymus during a specific developmental window yet are no longer detectable at birth. To analyze the function of the Lgr5 protein during thymus development, we generated Lgr5 −/− mice. These experiments unequivocally show that thymus development is not perturbed in the absence of Lgr5, that all TEC subsets develop in Lgr5 −/− mice and that T cells are produced in the expected ratios. Finally, by using an inducible lineage tracing system to track the progeny of Lgr5 + fetal TECs in vivo, we demonstrated that Lgr5 + fetal TECs have no detectable progeny in the later fetal thymus. In sum, we show that presence of the Lgr5 protein is not a prerequisite for proper thymus organogenesis. Keywords:Fetal thymus r Lgr5 r Lineage tracing r Thymic epithelial cells IntroductionThymic epithelial cells (TECs) form a 3D network that is essential for the proper proliferation, differentiation, and selection of developing thymocytes. Epithelial derived factors include growth factors, differentiation signals, and self-antigens expressed via MHC class I (MHCI) and MHC class II (MHCII) (reviewed in [1]). Presentation of self-antigens on TECs [2-5], the exact timing, affinity, and cofactors for the physiological contact between TECs and developing T cells (reviewed in [6]) is fundamental for proper T-cell development.The murine thymus originates from the third pharyngeal pouch at day E9.5 of embryonic development and is solely derived from the endoderm [7]. Specification of the thymus involves the sequential upregulation of important transcription factors (Hoxa3, Eya1, Rae2, chordin, and BMP; (reviewed in [8]) eventually leading to the expression of the thymic-specific transcription Correspondence: Prof. Jan J. Cornelissen e-mail: J.Cornelissen@erasmusmc.nl factor Foxn1 [9,10]. From E11.5 onwards, the first precursor T cells migrate into the thymic anlage and noncanonical NF-κB signaling becomes important for full differentiation of the medullary microenvironment, culminating in the upregulation of autoimmune regulator (Aire) [11][12][13] that enables medullary TECs to express self-antigens [2,3]. In the adult thymus cross-talk remains important, as the process of differentiation but also maintenance of medullary TECs, via ligation of RANK and CD40 by ligands expressed on thymocytes [11,12,14].Mature cortical and medullary TEC originate from a common thymic epithelial progenitor cell (TEPC) [15,16]. Although full differentiation of mature TECs from a clonal precursor population has been demonstrated, the precise phenotypical characterization of that precursor...

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