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Cell-autonomous defects in thymic epithelial cells disrupt endothelial-perivascular cell interactions in the mouse thymus.

Bryson JL, Griffith AV, Hughes B, Saito F, Takahama Y, Richie ER, Manley NR - PLoS ONE (2013)

Bottom Line: We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-β(+) mesenchymal cells following at E14.5.At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization.These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.

ABSTRACT
The thymus is composed of multiple stromal elements comprising specialized stromal microenvironments responsible for the development of self-tolerant and self-restricted T cells. Here, we investigated the ontogeny and maturation of the thymic vasculature. We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-β(+) mesenchymal cells following at E14.5. Using an allelic series of the thymic epithelial cell (TEC) specific transcription factor Foxn1, we showed that these events are delayed by 1-2 days in Foxn1 (Δ/Δ) mice, and this phenotype was exacerbated with reduced Foxn1 dosage. At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization. The expression of both VEGF-A and PDGF-B, which are both primarily expressed in vasculature-associated mesenchyme or endothelium in the thymus, were reduced at E13.5 and E15.5 in Foxn1 (Δ/Δ) mice compared with controls. These data suggest that Foxn1 is required in TECs both to recruit endothelial cells and for endothelial cells to communicate with thymic mesenchyme, and for the differentiation of vascular-associated mesenchymal cells. These data show that Foxn1 function in TECs is required for normal thymus size and to generate the cellular and molecular environment needed for normal thymic vascularization. These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.

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VEGF-A and PDGF-B expression reduced in Foxn1Δ/Δ thymus.(A) VEGF-A expression was significantly reduced in E13.5 Foxn1Δ/Δ(n = 4), E15.5 Foxn1Δ/Δ (n = 3), and E15.5 Foxn1Δ/nu (n = 6), compared to Foxn1+/Δ control thymi. PDGF-B expression was also reduced in E13.5 Foxn1Δ/Δ (n = 4), E15.5 Foxn1Δ/Δ (n = 3), and E15.5 Foxn1Δ/nu (n = 6), compared to Foxn1+/Δ control thymi. Experiments represent relative RNA expression of pooled thymi. Controls were set to 1. Asterisks denote statistical significance (P<0.05). (B–E) Immunofluorescence analysis of VEGF-A expression performed on frozen transverse sections of embryonic thymus for CD31+ (blue), VEGF-A (green) and Cytokeratin (red). VEGF-A expression was detected in thymic endothelium, perivascular cells, and TECs in Foxn1+/Δ and Foxn1Δ/Δ mice (B–E). VEGF-A expression was reduced in (D–E) E13.5 Foxn1Δ/Δ thymus compared to (B–C) E13.5 Foxn1+/Δ controls. Scale bar, 100 µm; n = 3 or more.
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pone-0065196-g008: VEGF-A and PDGF-B expression reduced in Foxn1Δ/Δ thymus.(A) VEGF-A expression was significantly reduced in E13.5 Foxn1Δ/Δ(n = 4), E15.5 Foxn1Δ/Δ (n = 3), and E15.5 Foxn1Δ/nu (n = 6), compared to Foxn1+/Δ control thymi. PDGF-B expression was also reduced in E13.5 Foxn1Δ/Δ (n = 4), E15.5 Foxn1Δ/Δ (n = 3), and E15.5 Foxn1Δ/nu (n = 6), compared to Foxn1+/Δ control thymi. Experiments represent relative RNA expression of pooled thymi. Controls were set to 1. Asterisks denote statistical significance (P<0.05). (B–E) Immunofluorescence analysis of VEGF-A expression performed on frozen transverse sections of embryonic thymus for CD31+ (blue), VEGF-A (green) and Cytokeratin (red). VEGF-A expression was detected in thymic endothelium, perivascular cells, and TECs in Foxn1+/Δ and Foxn1Δ/Δ mice (B–E). VEGF-A expression was reduced in (D–E) E13.5 Foxn1Δ/Δ thymus compared to (B–C) E13.5 Foxn1+/Δ controls. Scale bar, 100 µm; n = 3 or more.

Mentions: We next tested whether defects in Foxn1-dependent TEC differentiation affect VEGF-A and PDGF-B expression in the thymus. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular development during embryogenesis and in adults [37], [38], [39], [40]. In the thymus, VEGF-A expression has been reported in TECs, NCCs, endothelial cells, and a subset of immature thymocytes [19], [20]. We measured mRNA levels in thymic stromal cells of Foxn1Δ/Δ, Foxn1Δ/nu and control littermates. VEGF-A (Figure 8A) expression was significantly reduced (p<0.05) at both E13.5 and E15.5 in Foxn1Δ/Δ and at E15.5 Foxn1Δ/nu mice compared to control littermates. By immunofluorescence, VEGF-A protein was predominantly associated with vasculature, and TECs (Figure 8B–I). In E13.5 Foxn1Δ/Δ thymi, which do not have vasculature inside the thymus, VEGF-A protein staining was highest in the capsule vasculature, but virtually undetectable in TECs (Figure 8D–E). By E15.5, VEGF-A clearly delineates the vasculature in controls (Figure 8F–G) but not in Foxn1Δ/Δ thymi (Figure 8H–I), indicating that the decreased VEGF-A expression may be at least in part from the vascular-associated mesenchyme and/or endothelium.


Cell-autonomous defects in thymic epithelial cells disrupt endothelial-perivascular cell interactions in the mouse thymus.

Bryson JL, Griffith AV, Hughes B, Saito F, Takahama Y, Richie ER, Manley NR - PLoS ONE (2013)

VEGF-A and PDGF-B expression reduced in Foxn1Δ/Δ thymus.(A) VEGF-A expression was significantly reduced in E13.5 Foxn1Δ/Δ(n = 4), E15.5 Foxn1Δ/Δ (n = 3), and E15.5 Foxn1Δ/nu (n = 6), compared to Foxn1+/Δ control thymi. PDGF-B expression was also reduced in E13.5 Foxn1Δ/Δ (n = 4), E15.5 Foxn1Δ/Δ (n = 3), and E15.5 Foxn1Δ/nu (n = 6), compared to Foxn1+/Δ control thymi. Experiments represent relative RNA expression of pooled thymi. Controls were set to 1. Asterisks denote statistical significance (P<0.05). (B–E) Immunofluorescence analysis of VEGF-A expression performed on frozen transverse sections of embryonic thymus for CD31+ (blue), VEGF-A (green) and Cytokeratin (red). VEGF-A expression was detected in thymic endothelium, perivascular cells, and TECs in Foxn1+/Δ and Foxn1Δ/Δ mice (B–E). VEGF-A expression was reduced in (D–E) E13.5 Foxn1Δ/Δ thymus compared to (B–C) E13.5 Foxn1+/Δ controls. Scale bar, 100 µm; n = 3 or more.
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pone-0065196-g008: VEGF-A and PDGF-B expression reduced in Foxn1Δ/Δ thymus.(A) VEGF-A expression was significantly reduced in E13.5 Foxn1Δ/Δ(n = 4), E15.5 Foxn1Δ/Δ (n = 3), and E15.5 Foxn1Δ/nu (n = 6), compared to Foxn1+/Δ control thymi. PDGF-B expression was also reduced in E13.5 Foxn1Δ/Δ (n = 4), E15.5 Foxn1Δ/Δ (n = 3), and E15.5 Foxn1Δ/nu (n = 6), compared to Foxn1+/Δ control thymi. Experiments represent relative RNA expression of pooled thymi. Controls were set to 1. Asterisks denote statistical significance (P<0.05). (B–E) Immunofluorescence analysis of VEGF-A expression performed on frozen transverse sections of embryonic thymus for CD31+ (blue), VEGF-A (green) and Cytokeratin (red). VEGF-A expression was detected in thymic endothelium, perivascular cells, and TECs in Foxn1+/Δ and Foxn1Δ/Δ mice (B–E). VEGF-A expression was reduced in (D–E) E13.5 Foxn1Δ/Δ thymus compared to (B–C) E13.5 Foxn1+/Δ controls. Scale bar, 100 µm; n = 3 or more.
Mentions: We next tested whether defects in Foxn1-dependent TEC differentiation affect VEGF-A and PDGF-B expression in the thymus. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular development during embryogenesis and in adults [37], [38], [39], [40]. In the thymus, VEGF-A expression has been reported in TECs, NCCs, endothelial cells, and a subset of immature thymocytes [19], [20]. We measured mRNA levels in thymic stromal cells of Foxn1Δ/Δ, Foxn1Δ/nu and control littermates. VEGF-A (Figure 8A) expression was significantly reduced (p<0.05) at both E13.5 and E15.5 in Foxn1Δ/Δ and at E15.5 Foxn1Δ/nu mice compared to control littermates. By immunofluorescence, VEGF-A protein was predominantly associated with vasculature, and TECs (Figure 8B–I). In E13.5 Foxn1Δ/Δ thymi, which do not have vasculature inside the thymus, VEGF-A protein staining was highest in the capsule vasculature, but virtually undetectable in TECs (Figure 8D–E). By E15.5, VEGF-A clearly delineates the vasculature in controls (Figure 8F–G) but not in Foxn1Δ/Δ thymi (Figure 8H–I), indicating that the decreased VEGF-A expression may be at least in part from the vascular-associated mesenchyme and/or endothelium.

Bottom Line: We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-β(+) mesenchymal cells following at E14.5.At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization.These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.

ABSTRACT
The thymus is composed of multiple stromal elements comprising specialized stromal microenvironments responsible for the development of self-tolerant and self-restricted T cells. Here, we investigated the ontogeny and maturation of the thymic vasculature. We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-β(+) mesenchymal cells following at E14.5. Using an allelic series of the thymic epithelial cell (TEC) specific transcription factor Foxn1, we showed that these events are delayed by 1-2 days in Foxn1 (Δ/Δ) mice, and this phenotype was exacerbated with reduced Foxn1 dosage. At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization. The expression of both VEGF-A and PDGF-B, which are both primarily expressed in vasculature-associated mesenchyme or endothelium in the thymus, were reduced at E13.5 and E15.5 in Foxn1 (Δ/Δ) mice compared with controls. These data suggest that Foxn1 is required in TECs both to recruit endothelial cells and for endothelial cells to communicate with thymic mesenchyme, and for the differentiation of vascular-associated mesenchymal cells. These data show that Foxn1 function in TECs is required for normal thymus size and to generate the cellular and molecular environment needed for normal thymic vascularization. These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.

Show MeSH
Related in: MedlinePlus