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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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Peripheral circulation is connected to the thymus at E14.5.FITC-dextran (green) facial vein injections and immunostaining for CD31 (red) and cytokeratin (blue) on frozen sagittal sections of fetal mouse thymus. (A–B) FITC-dextran is detected in E14.5 Foxn1+/Δ thymi, tightly associated with CD31+ blood vessels. (C–D) In Foxn1Δ/Δ embryos, FITC-dextran is also present, but the signal is more diffusely associated with vessels. (E–F) FITC-dextran is present throughout E18.5 Foxn1+/Δ thymi tightly associated with branched blood vessels. (G–H) FITC-dextran is more diffusely present in the thymus of E18.5 Foxn1Δ/Δ mice. Scale bar, 100 µm; n = 3.
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pone-0065196-g005: Peripheral circulation is connected to the thymus at E14.5.FITC-dextran (green) facial vein injections and immunostaining for CD31 (red) and cytokeratin (blue) on frozen sagittal sections of fetal mouse thymus. (A–B) FITC-dextran is detected in E14.5 Foxn1+/Δ thymi, tightly associated with CD31+ blood vessels. (C–D) In Foxn1Δ/Δ embryos, FITC-dextran is also present, but the signal is more diffusely associated with vessels. (E–F) FITC-dextran is present throughout E18.5 Foxn1+/Δ thymi tightly associated with branched blood vessels. (G–H) FITC-dextran is more diffusely present in the thymus of E18.5 Foxn1Δ/Δ mice. Scale bar, 100 µm; n = 3.

Mentions: A critical event during thymic organogenesis is the connection of the peripheral vasculature and circulation to the developing vessels in the thymic anlage. This developmental time point likely indicates a switch in the route of LPC entry into the thymus, from initial trans-capsular migration to vascular extravasation at late fetal and postnatal stages [6], [22], [25], [36]. As endothelial cells initially immigrate into the fetal thymus at E13.5, the external connection must occur after this point. To identify the developmental time point at which the peripheral circulation connects to the thymus, we performed FITC-dextran facial vein injections at E14.5. FITC-dextran was detected in Foxn1+/Δ thymi tightly associated with CD31+ vasculature (Figure 5A–B). The timing of intrathymic FITC-dextran detection was similar in Foxn1Δ/Δ mutants (Figure 5C–D), although fewer vessels were labeled in the mutants, and the association of dye with vascular cells appeared looser. This result was consistent with previous data demonstrating the connection is established by E15.5 [14], and suggested that timing of this connection to the embryonic vasculature was regulated by Foxn1-independent mechanisms.


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)

Peripheral circulation is connected to the thymus at E14.5.FITC-dextran (green) facial vein injections and immunostaining for CD31 (red) and cytokeratin (blue) on frozen sagittal sections of fetal mouse thymus. (A–B) FITC-dextran is detected in E14.5 Foxn1+/Δ thymi, tightly associated with CD31+ blood vessels. (C–D) In Foxn1Δ/Δ embryos, FITC-dextran is also present, but the signal is more diffusely associated with vessels. (E–F) FITC-dextran is present throughout E18.5 Foxn1+/Δ thymi tightly associated with branched blood vessels. (G–H) FITC-dextran is more diffusely present in the thymus of E18.5 Foxn1Δ/Δ mice. Scale bar, 100 µm; n = 3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672159&req=5

pone-0065196-g005: Peripheral circulation is connected to the thymus at E14.5.FITC-dextran (green) facial vein injections and immunostaining for CD31 (red) and cytokeratin (blue) on frozen sagittal sections of fetal mouse thymus. (A–B) FITC-dextran is detected in E14.5 Foxn1+/Δ thymi, tightly associated with CD31+ blood vessels. (C–D) In Foxn1Δ/Δ embryos, FITC-dextran is also present, but the signal is more diffusely associated with vessels. (E–F) FITC-dextran is present throughout E18.5 Foxn1+/Δ thymi tightly associated with branched blood vessels. (G–H) FITC-dextran is more diffusely present in the thymus of E18.5 Foxn1Δ/Δ mice. Scale bar, 100 µm; n = 3.
Mentions: A critical event during thymic organogenesis is the connection of the peripheral vasculature and circulation to the developing vessels in the thymic anlage. This developmental time point likely indicates a switch in the route of LPC entry into the thymus, from initial trans-capsular migration to vascular extravasation at late fetal and postnatal stages [6], [22], [25], [36]. As endothelial cells initially immigrate into the fetal thymus at E13.5, the external connection must occur after this point. To identify the developmental time point at which the peripheral circulation connects to the thymus, we performed FITC-dextran facial vein injections at E14.5. FITC-dextran was detected in Foxn1+/Δ thymi tightly associated with CD31+ vasculature (Figure 5A–B). The timing of intrathymic FITC-dextran detection was similar in Foxn1Δ/Δ mutants (Figure 5C–D), although fewer vessels were labeled in the mutants, and the association of dye with vascular cells appeared looser. This result was consistent with previous data demonstrating the connection is established by E15.5 [14], and suggested that timing of this connection to the embryonic vasculature was regulated by Foxn1-independent mechanisms.

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