Differential requirement for mesenchyme in the proliferation and maturation of thymic epithelial progenitors - PubMed (original) (raw)

Differential requirement for mesenchyme in the proliferation and maturation of thymic epithelial progenitors

William E Jenkinson et al. J Exp Med. 2003.

Abstract

Formation of a mature thymic epithelial microenvironment is an essential prerequisite for the generation of a functionally competent T cell pool. It is likely that recently identified thymic epithelial precursors undergo phases of proliferation and differentiation to generate mature cortical and medullary thymic microenvironments. The mechanisms regulating development of immature thymic epithelial cells are unknown. Here we provide evidence that expansion of embryonic thymic epithelium is regulated by the continued presence of mesenchyme. In particular, mesenchymal cells are shown to mediate thymic epithelial cell proliferation through their provision of fibroblast growth factors 7 and 10. In contrast, differentiation of immature thymic epithelial cells, including acquisition of markers of mature cortical and medullary epithelium, occurs in the absence of ongoing mesenchymal support. Collectively, our data define a role for mesenchymal cells in thymus development, and indicate distinct mechanisms regulate proliferation and differentiation of immature thymic epithelial cells. In addition, our findings may aid in studies aimed at developing strategies to enhance thymus reconstitution and functioning in clinical certain contexts where thymic epithelial cell function is perturbed.

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Figures

Figure 1.

Mesenchyme and not thymocytes regulate proliferation of embryonic thymic epithelium. Proliferating thymic epithelial cells were identified in fetal thymus lobes by analyzing cytokeratin expression in combination with BrdU incorporation. Panel a shows a typical example of flow cytometric analysis of E14 fetal thymus lobes, where a Keratin(K)+BrdU+ population is clearly visible. Panel c shows analysis of epithelial cell proliferation in wild-type and CD3ɛtg26 E14 thymus lobes after an 18 h BrdU pulse. The percentage of K+ epithelial cells incorporating BrdU after an 18 h pulse was also analyzed in whole E12 and E14 thymus lobes, and in E12 and E14 thymuses devoid of mesenchyme (d). Note reduction in epithelial proliferation at both developmental stages in the absence mesenchyme. In all flow cytometric analyses, a minimum of 10,000 gated events was analyzed. Panel b shows E12 thymic lobes with surrounding perithymic mesenchyme attached (arrow) and after enzymatic removal of perithymic mesenchyme to leave an epithelial thymic rudiment (arrowhead). Similar results were obtained from at least three separate experiments.

Figure 2.

Mesenchymal production of FGF7 and FGF10 regulate proliferation of embryonic thymic epithelium. Semiquantitative PCR (a) for FGF7, FGF10, and FGFR2IIIb was performed on cDNAs isolated from thymic epithelium, thymic mesenchyme and CD4−8− thymocytes purified from E14 thymus. Equal loading of cDNA was monitored by analyzing β-actin mRNA expression. (b) Proliferation of thymic epithelium was analyzed by BrdU incorporation and Keratin expression in reaggregate cultures formed from whole E14 lobes, and from purified E14 thymic epithelium reaggregate cultures, the latter being cultured in the presence or absence of 100 ng/ml FGF7 and FGF10, either singularly or in combination. Experiments were performed three times with similar results.

Figure 3.

Embryonic day 12 thymic epithelial cells acquire K5+8− and K5−8+ phenotypes in the absence of mesenchyme. Cytospins were prepared from E12 thymus (a), and E12 thymus lobes cultured for 2 d with either mesenchyme attached (b) or removed (c). Cytospins were stained with antibodies to keratin 5 (red) and keratin 8 (green), with K5+8+ cells visible where fluorochromes overlap to give yellow (arrowed). Frequencies of K5+8+, K5−8+, and K5+8− subsets (d) was determined by counting at least 100 cells per experiment using a fluorescent microscope. Data are representative of three separate experiments.

Figure 4.

Phenotypic and genotypic differentiation of E12 thymic epithelium in the absence of mesenchyme. E12 fetal thymus lobes were organ cultured intact or after enzymatic removal of mesenchyme for either 2 d (for RT-PCR analysis) or 6 d (for flow cytometric analysis). For RT-PCR analysis (a), cDNAs were used to determine expression of Aire and Plunc, with equal cDNA loadings monitored by β-actin levels. For flow cytometry (b), 6 d cultures were disaggregated and analyzed for cell surface MHC class II expression. Data shown are representative of three separate experiments.

Figure 5.

E12 thymic epithelial cells grown in the absence of mesenchyme are functionally mature. Freshly purified CD4+8+ thymocytes, obtained from neonatal thymus lobes, were reaggregated at 1:1 mixtures with either 2-dGuo treated stroma (a), or epithelial cells from E12 thymus lobes cultured in the absence of mesenchyme for 7 d (b). After 6 d of reaggregate culture, thymocytes were harvested and stained for CD4 and CD8. Note the presence of CD4+8− and CD4−8+ cells in both cultures. In the experiment shown, 105 epithelial cells were reaggregated with 2 × 105 stromal cells, with yields of 2.5 × 104 and 2 × 104 from cultures of 2-deoxyguanosine and cultured E12 epithelium, respectively. Data shown are representative of three separate experiments.

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Differential requirement for mesenchyme in the proliferation and maturation of thymic epithelial progenitors - PubMed (original) (raw)