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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 embryonic thymic vascularization is defective in Foxn1Δ/Δ Mice.Immunostaining on frozen transverse sections of fetal thymus (E12.5–E14.5). Endothelial and stromal cell markers used are listed above each column in the corresponding color: CD31+/CD144+ for endothelial cells (green); PDGFR-β+ for neural crest mesenchyme (red); Cytokeratin (blue) or Keratin 5 (red, K5) for epithelial cells. Embryonic stages in the first column and genotypes to the left apply to the entire row unless otherwise labeled. (A–e) CD31+/CD144+ endothelial cells and PDGFR-β+ neural crest cells are present in the thymic capsule region in Foxn1+/Δ (A–E) and Foxn1Δ/Δ mice (a–e) at E12.5. (F–o) CD31+/CD144+ cells followed by PDGFR-β+ cells initially immigrate into the thymus at E13.5 in heterozygotes (F–J) and at E14.5 in homozygotes (k–o). Scale bar, 50 µm; n = 3.
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pone-0065196-g002: Initial embryonic thymic vascularization is defective in Foxn1Δ/Δ Mice.Immunostaining on frozen transverse sections of fetal thymus (E12.5–E14.5). Endothelial and stromal cell markers used are listed above each column in the corresponding color: CD31+/CD144+ for endothelial cells (green); PDGFR-β+ for neural crest mesenchyme (red); Cytokeratin (blue) or Keratin 5 (red, K5) for epithelial cells. Embryonic stages in the first column and genotypes to the left apply to the entire row unless otherwise labeled. (A–e) CD31+/CD144+ endothelial cells and PDGFR-β+ neural crest cells are present in the thymic capsule region in Foxn1+/Δ (A–E) and Foxn1Δ/Δ mice (a–e) at E12.5. (F–o) CD31+/CD144+ cells followed by PDGFR-β+ cells initially immigrate into the thymus at E13.5 in heterozygotes (F–J) and at E14.5 in homozygotes (k–o). Scale bar, 50 µm; n = 3.

Mentions: To identify initial stages of thymus vascularization, we first performed a histological analysis of the embryonic thymus and assayed for the presence of morphologically apparent blood vessels containing red blood cells (RBCs) in the rudiment (Figure 1). Vessels containing RBC were detected as early as E13.5 in the Foxn1+/Δ thymus (Figure 1A), but were noticeably absent in Foxn1Δ/Δ littermates (Figure 1B). RBCs were detected in both E14.5 Foxn1+/Δ and Foxn1Δ/Δ thymi (Figure 1C–D). We next assayed E12.5–E14.5 Foxn1+/Δ and Foxn1Δ/Δ embryonic thymi for the presence of two endothelial cell markers, PECAM-1 (CD31) and VE-Cadherin (CD144). We also confirmed that the timing of vascularization events was similar between wild-type and Foxn1+/Δ embryos (Figure S2), confirming that the heterozygotes were appropriate controls for these events. CD31+ and CD144+ endothelial cells surrounded the thymic epithelial rudiment by E12.5 (Figure 2A, D–E, and 3A), just outside of the PDGFRβ+ neural crest-derived mesenchymal capsule that encompasses the thymic rudiment [13], [16], [17], [18]. By E13.5, a network of nascent vascular endothelial structures were present within the thymus and associated with the centrally localized Keratin 5+ (K5) subset of thymic epithelial cells (Figure 2F, I–J and 3A). Endothelial cells also remained associated with PDGFR-β+ cells in the perithymic mesenchyme (Figure 2F, G, and I) [14]. At E14.5, the vascular network was markedly more complex, with an increased number of CD31+ and CD144+ cells within the thymus (Figure 2K, N–O and 3B–D). At this stage PDGFR-β+ cells had migrated into the thymus in close association with the vascular network (Figure 2N). PGDFR-β+ cells in the capsule also began to migrate into the TEC network at E14.5 (Figure 2L, N and 3C–D). These data are consistent with a model in which PDGFR-β+ mesenchyme and endothelial cells first encapsulate the E12.5 thymus, and that vascular branches subsequently penetrate the thymus capsule, followed by PDGFR-β+ mesenchymal cells migrating along the endothelium into the thymic rudiment.


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 embryonic thymic vascularization is defective in Foxn1Δ/Δ Mice.Immunostaining on frozen transverse sections of fetal thymus (E12.5–E14.5). Endothelial and stromal cell markers used are listed above each column in the corresponding color: CD31+/CD144+ for endothelial cells (green); PDGFR-β+ for neural crest mesenchyme (red); Cytokeratin (blue) or Keratin 5 (red, K5) for epithelial cells. Embryonic stages in the first column and genotypes to the left apply to the entire row unless otherwise labeled. (A–e) CD31+/CD144+ endothelial cells and PDGFR-β+ neural crest cells are present in the thymic capsule region in Foxn1+/Δ (A–E) and Foxn1Δ/Δ mice (a–e) at E12.5. (F–o) CD31+/CD144+ cells followed by PDGFR-β+ cells initially immigrate into the thymus at E13.5 in heterozygotes (F–J) and at E14.5 in homozygotes (k–o). Scale bar, 50 µm; n = 3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672159&req=5

pone-0065196-g002: Initial embryonic thymic vascularization is defective in Foxn1Δ/Δ Mice.Immunostaining on frozen transverse sections of fetal thymus (E12.5–E14.5). Endothelial and stromal cell markers used are listed above each column in the corresponding color: CD31+/CD144+ for endothelial cells (green); PDGFR-β+ for neural crest mesenchyme (red); Cytokeratin (blue) or Keratin 5 (red, K5) for epithelial cells. Embryonic stages in the first column and genotypes to the left apply to the entire row unless otherwise labeled. (A–e) CD31+/CD144+ endothelial cells and PDGFR-β+ neural crest cells are present in the thymic capsule region in Foxn1+/Δ (A–E) and Foxn1Δ/Δ mice (a–e) at E12.5. (F–o) CD31+/CD144+ cells followed by PDGFR-β+ cells initially immigrate into the thymus at E13.5 in heterozygotes (F–J) and at E14.5 in homozygotes (k–o). Scale bar, 50 µm; n = 3.
Mentions: To identify initial stages of thymus vascularization, we first performed a histological analysis of the embryonic thymus and assayed for the presence of morphologically apparent blood vessels containing red blood cells (RBCs) in the rudiment (Figure 1). Vessels containing RBC were detected as early as E13.5 in the Foxn1+/Δ thymus (Figure 1A), but were noticeably absent in Foxn1Δ/Δ littermates (Figure 1B). RBCs were detected in both E14.5 Foxn1+/Δ and Foxn1Δ/Δ thymi (Figure 1C–D). We next assayed E12.5–E14.5 Foxn1+/Δ and Foxn1Δ/Δ embryonic thymi for the presence of two endothelial cell markers, PECAM-1 (CD31) and VE-Cadherin (CD144). We also confirmed that the timing of vascularization events was similar between wild-type and Foxn1+/Δ embryos (Figure S2), confirming that the heterozygotes were appropriate controls for these events. CD31+ and CD144+ endothelial cells surrounded the thymic epithelial rudiment by E12.5 (Figure 2A, D–E, and 3A), just outside of the PDGFRβ+ neural crest-derived mesenchymal capsule that encompasses the thymic rudiment [13], [16], [17], [18]. By E13.5, a network of nascent vascular endothelial structures were present within the thymus and associated with the centrally localized Keratin 5+ (K5) subset of thymic epithelial cells (Figure 2F, I–J and 3A). Endothelial cells also remained associated with PDGFR-β+ cells in the perithymic mesenchyme (Figure 2F, G, and I) [14]. At E14.5, the vascular network was markedly more complex, with an increased number of CD31+ and CD144+ cells within the thymus (Figure 2K, N–O and 3B–D). At this stage PDGFR-β+ cells had migrated into the thymus in close association with the vascular network (Figure 2N). PGDFR-β+ cells in the capsule also began to migrate into the TEC network at E14.5 (Figure 2L, N and 3C–D). These data are consistent with a model in which PDGFR-β+ mesenchyme and endothelial cells first encapsulate the E12.5 thymus, and that vascular branches subsequently penetrate the thymus capsule, followed by PDGFR-β+ mesenchymal cells migrating along the endothelium into the thymic rudiment.

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