ABSTRACT
The study of organogenesis, tissue-homeostasis and regeneration requires the precise assessment of in vivo cell proliferation. To this end a host of methods have been developed to detect and quantify DNA synthesis in proliferating cells. These include cell labeling with various nucleotide analogues and fluorescence reporter-based animal models with each method presenting its idiosyncratic shortcomings. Quantitative assessment of epithelial cell turnover has been partly hampered due to their variable and limited in vivo accessibility and the requirement for harsher isolation procedures to procure single cells for FACS analysis. Here, we report a reliable protocol to study in vivo cell proliferation of epithelial cells in mice by repeatedly injecting EdU intravenously for an extended 12-day period. EdU incorporation was quantitated ex vivo by FACS after tissue dissociation in order to obtain single epithelial cell suspensions. As a lead population, we analyzed thymic epithelial cells (TECs), where we were able to label compartmentalized TEC subsets to saturation without apparent toxic effects on the thymus architecture or stress-sensitive TEC lineage differentiation. The data is in concordance with the prevailing model of medullary TEC terminal differentiation that includes the post-Aire stage. The same protocol was successfully applied to epithelial cells of various other organs - skin, lymph node, kidney and small intestine - tissues with widely varying frequencies and rates of proliferating epithelial cells.
Subject(s)
Cell Differentiation , Cell Proliferation , Epithelial Cells/cytology , Staining and Labeling/methods , Administration, Intravenous , Animals , Cell Count , Deoxyuridine/administration & dosage , Deoxyuridine/analogs & derivatives , Flow Cytometry , Mice , Mice, Inbred C57BL , Thymus Gland/cytologyABSTRACT
The basic two-step terminal differentiation model of the medullary thymic epithelial cell (mTEC) lineage from immature MHC class II (MHCII)lo to mature MHCIIhi mTECs has recently been extended to include a third stage, namely the post-Aire MHCIIlo subset as identified by lineage-tracing models. However, a suitable surface marker distinguishing the phenotypically overlapping pre- from the post-Aire MHCIIlo stage has been lacking. In this study, we introduce the lectin Tetragonolobus purpureas agglutinin (TPA) as a novel cell surface marker that allows for such delineation. Based on our data, we derived the following sequence of mTEC differentiation: TPAloMHCIIlo â TPAloMHCIIhi â TPAhiMHCIIhi â TPAhiMHCIIlo Surprisingly, in the steady-state postnatal thymus TPAloMHCIIlo pre-Aire rather than terminally differentiated post-Aire TPAhiMHCIIlo mTECs were marked for apoptosis at an exceptionally high rate of â¼70%. Hence, only the minor cycling fraction of the MHCIIlo subset (<20%) potentially qualified as mTEC precursors. FoxN1 expression inversely correlated with the fraction of slow cycling and apoptotic cells within the four TPA subsets. TPA also further subdivided human mTECs, although with different subset distribution. Our revised road map emphazises close parallels of terminal mTEC development with that of skin, undergoing an alternative route of cell death, namely cornification rather than apoptosis. The high rate of apoptosis in pre-Aire MHCIIlo mTECs points to a "quality control" step during early mTEC differentiation.
Subject(s)
Biomarkers/metabolism , Epithelial Cells/physiology , Lectins/metabolism , Thymus Gland/cytology , Transcription Factors/metabolism , Animals , Apoptosis , Cell Differentiation , Cell Lineage , Cells, Cultured , Female , Gene Expression Regulation , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Pregnancy , Rats , Rats, Sprague-Dawley , Thymus Gland/anatomy & histology , Transcription Factors/genetics , AIRE ProteinABSTRACT
Expression of tissue-restricted self antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for the induction of self-tolerance and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and is coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA sequencing and obtained evidence of numerous recurring TRA-co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process that might involve local remodeling of chromatin and thus ensures a comprehensive representation of the immunological self.
