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FoxN1 mediates thymic cortex–medulla differentiation through modifying a developmental pattern based on epithelial tubulogenesis

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Abstract

The mechanisms that determine the commitment of thymic epithelial precursors to the two major thymic epithelial cell lineages, cTECs and mTECs, remain unknown. Here we show that FoxN1 nu mutation, which abolishes thymic epithelium differentiation, results in the formation of a tubular branched structure according to a typical branching morphogenesis and tubulogenesis developmental pattern. In the presence of FoxN1, in alymphoid NSG and fetal Ikaros−/− thymi, there is no lumen formation and only partial apical differentiation. This initiates cortex–medulla differentiation inducing expression of medullary genes in the apically differentiating cells and of cortical genes in the non-apically differentiating cells, which will definitely differentiate in wt and postnatal Ikaros−/− mice. Therefore, the thymus development is based on a branching morphogenesis and tubulogenesis developmental pattern: FoxN1 expression in the thymic primordium inhibits tubulogenesis and induces the expression of genes involved in TEC differentiation, which culminates with the expression of functional cell markers, i.e., MHCII, CD80, Aire in both postnatal Ikaros−/− and WT thymi after arrival of lymphoid progenitor cells.

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Acknowledgements

This work was supported by Grants BFU2013-41112-R and Cell Therapy Network (RD 16/0011/0002) from the Spanish Ministry of Economy and Competitiveness. We thank the Cytometry and Fluorescence Microscopy Centre of Complutense University for the use of its facilities. We thank the “Developmental Studies Hybridoma Bank” of the University of Iowa and Monash University Medical Centre, Melbourne, Australia for supplying anti-K8 keratin and anti-MTS10 antibodies, respectively. We also thank Dr. Josep Maria Canals (Barcelona University) for providing Ikaros−/− mice. Finally, we thank Dr. Nuno L. Alves (IBMC, Porto) for his critical reading of the manuscript and comments.

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  1. Esther Tobajas

    Present address: Beacon Biomedicine, Science To Business, Tres Cantos, 28760, Madrid, Spain

Authors and Affiliations

  1. Centre for Cytometry and Fluorescence Microscopy UCM, Complutense University of Madrid, 28040, Madrid, Spain

    Juan J. Muñoz, Sonia Juara & Agustín G. Zapata

  2. Department of Cell Biology, Faculty of Biology, Complutense University of Madrid, 28040, Madrid, Spain

    Esther Tobajas, Sara Montero & Agustín G. Zapata

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  1. Juan J. Muñoz
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418_2019_1818_MOESM1_ESM.tif

Supplementary material 1. Figure S1 Proliferation of nude thymic epithelial cells throughout development. Sections from thymic lobes of the indicated ages were stained for E-cad, Cld3/4, Ki67 and nuclear staining (Hoechst 33342). Cld+ and Cld− regions were segmented as shown in the examples. Nuclei within each region were segmented and counted as in the examples. Note the distribution of proliferating cells through the whole epithelial mass, the absence of proliferating tips and, also, the lower proportion of proliferating cells in E18 and 3PN thymic sections. Scale bar = 50 μm E13.5–E15.5; = 100 μm E18.5, 3PN; = 150 μm 3PN (TIFF 280221 kb)

418_2019_1818_MOESM2_ESM.tif

Supplementary material 2. Figure S2 FoxN1 expression in NSG thymus inhibits ZO1 expression and tight junction organization. Section from 15 days postnatal nude thymus and 6 days postnatal NSG thymus were stained for ZO1, E-cad and Cld3/4. Merge images are shown. Note the arrangement of ZO1 expression in tight junctions in nude thymus. In NSG thymus, ZO1 expression is found in some thymic cysts (white arrows) and in blood vessels (yellow arrows) while no expression or traces are found in most medullary areas (arrowheads) where nonplorarized Cld3/4 expression is found (TIFF 172494 kb)

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Muñoz, J.J., Tobajas, E., Juara, S. et al. FoxN1 mediates thymic cortex–medulla differentiation through modifying a developmental pattern based on epithelial tubulogenesis. Histochem Cell Biol 152, 397–413 (2019). https://doi.org/10.1007/s00418-019-01818-z

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