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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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Initial LPC thymic immigration is normal in Foxn1Δ mice.(A) CD45+ LPCs (green) colonize the Foxn1+/Δ and (B) Foxn1Δ/Δ thymus at E11.5. (C) At E11.5, the frequency of CD45+ cells/section was similar between Foxn1+/Δ (n = 10) and Foxn1Δ/Δ (n = 9) thymi; (p>0.05). (D) Immunostaining for CCL21 (green) expression is similar in Foxn1+/Δ and (E) Foxn1Δ/Δ mouse thymus. (F–I) Reduced expression of CCL25 (white) in (G and I) Foxn1Δ/Δ compared to (F and H) Foxn1+/Δ thymus at E11.5. Cytokeratin (red). (J) CCL25 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. CD45+ cells (green) were noticeably reduced in (K–L) E12.5, (M–N) E13.5, and (O–P) E14.5 Foxn1Δ/Δ thymi compared to control littermates. Scale bar, 100 µm. qRT experiments represent relative RNA expression of pooled thymi. Controls were set to 1. Asterisks denote statistical significance.
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pone-0065196-g004: Initial LPC thymic immigration is normal in Foxn1Δ mice.(A) CD45+ LPCs (green) colonize the Foxn1+/Δ and (B) Foxn1Δ/Δ thymus at E11.5. (C) At E11.5, the frequency of CD45+ cells/section was similar between Foxn1+/Δ (n = 10) and Foxn1Δ/Δ (n = 9) thymi; (p>0.05). (D) Immunostaining for CCL21 (green) expression is similar in Foxn1+/Δ and (E) Foxn1Δ/Δ mouse thymus. (F–I) Reduced expression of CCL25 (white) in (G and I) Foxn1Δ/Δ compared to (F and H) Foxn1+/Δ thymus at E11.5. Cytokeratin (red). (J) CCL25 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. CD45+ cells (green) were noticeably reduced in (K–L) E12.5, (M–N) E13.5, and (O–P) E14.5 Foxn1Δ/Δ thymi compared to control littermates. Scale bar, 100 µm. qRT experiments represent relative RNA expression of pooled thymi. Controls were set to 1. Asterisks denote statistical significance.

Mentions: We previously reported that Foxn1Δ/Δ mice have a significant decrease in total thymocytes at both fetal and adult stages [27]. In nude mice (Foxn1nu/nu), bone marrow derived-hematopoietic precursor cells (HPC) migrate to, but fail to colonize the thymus rudiment [14]. We therefore tested whether the Foxn1Δ mutation affected the timing of initial HPC infiltration of the thymus, which might contribute to these early vascular defects in Foxn1Δ/Δ mice. At E11.5, when thymocytes initially infiltrate the thymus [22], CD45+ cells were present in the thymus rudiment in both Foxn1Δ/Δ and control littermates at similar frequencies (Figure 4A–B and C). At E12.5–E14.5, CD45+ cells were significantly reduced in the mutant thymus, consistent with our previous results [27](Figure 4K–P). These data suggest that although CD45+ cells in the thymus are reduced after E11.5, the timing of initial HPC colonization of the thymus is normal in Foxn1Δ/Δ mice.


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)

Initial LPC thymic immigration is normal in Foxn1Δ mice.(A) CD45+ LPCs (green) colonize the Foxn1+/Δ and (B) Foxn1Δ/Δ thymus at E11.5. (C) At E11.5, the frequency of CD45+ cells/section was similar between Foxn1+/Δ (n = 10) and Foxn1Δ/Δ (n = 9) thymi; (p>0.05). (D) Immunostaining for CCL21 (green) expression is similar in Foxn1+/Δ and (E) Foxn1Δ/Δ mouse thymus. (F–I) Reduced expression of CCL25 (white) in (G and I) Foxn1Δ/Δ compared to (F and H) Foxn1+/Δ thymus at E11.5. Cytokeratin (red). (J) CCL25 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. CD45+ cells (green) were noticeably reduced in (K–L) E12.5, (M–N) E13.5, and (O–P) E14.5 Foxn1Δ/Δ thymi compared to control littermates. Scale bar, 100 µm. qRT experiments represent relative RNA expression of pooled thymi. Controls were set to 1. Asterisks denote statistical significance.
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Related In: Results  -  Collection

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pone-0065196-g004: Initial LPC thymic immigration is normal in Foxn1Δ mice.(A) CD45+ LPCs (green) colonize the Foxn1+/Δ and (B) Foxn1Δ/Δ thymus at E11.5. (C) At E11.5, the frequency of CD45+ cells/section was similar between Foxn1+/Δ (n = 10) and Foxn1Δ/Δ (n = 9) thymi; (p>0.05). (D) Immunostaining for CCL21 (green) expression is similar in Foxn1+/Δ and (E) Foxn1Δ/Δ mouse thymus. (F–I) Reduced expression of CCL25 (white) in (G and I) Foxn1Δ/Δ compared to (F and H) Foxn1+/Δ thymus at E11.5. Cytokeratin (red). (J) CCL25 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. CD45+ cells (green) were noticeably reduced in (K–L) E12.5, (M–N) E13.5, and (O–P) E14.5 Foxn1Δ/Δ thymi compared to control littermates. Scale bar, 100 µm. qRT experiments represent relative RNA expression of pooled thymi. Controls were set to 1. Asterisks denote statistical significance.
Mentions: We previously reported that Foxn1Δ/Δ mice have a significant decrease in total thymocytes at both fetal and adult stages [27]. In nude mice (Foxn1nu/nu), bone marrow derived-hematopoietic precursor cells (HPC) migrate to, but fail to colonize the thymus rudiment [14]. We therefore tested whether the Foxn1Δ mutation affected the timing of initial HPC infiltration of the thymus, which might contribute to these early vascular defects in Foxn1Δ/Δ mice. At E11.5, when thymocytes initially infiltrate the thymus [22], CD45+ cells were present in the thymus rudiment in both Foxn1Δ/Δ and control littermates at similar frequencies (Figure 4A–B and C). At E12.5–E14.5, CD45+ cells were significantly reduced in the mutant thymus, consistent with our previous results [27](Figure 4K–P). These data suggest that although CD45+ cells in the thymus are reduced after E11.5, the timing of initial HPC colonization of the thymus is normal in Foxn1Δ/Δ mice.

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