Figure 1.
Histological Analysis of vasculature in embryonic thymus of Foxn1Δ/Δ Mice.
Haematoxylin & Eosin staining on paraffin section of fetal thymus (E13.5–E14.5). (A) E13.5 Foxn1+/Δ embryonic thymus with RBC detected throughout rudiment (B) Absence of RBC in E13.5 Foxn1Δ/Δ embryonic thymus (C) RBC present in E14.5 Foxn1+/Δ and (D) Foxn1Δ/Δ embryonic thymus. Scale bar = 50 µm; n = 3.
Figure 2.
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.
Figure 3.
Thymic vascularization is sensitive to Foxn1 levels.
Quantification of CD31+/Thymus Area from immunostained frozen sections of embryonic thymi (A) E12.5 Foxn1+/Δ (n = 8) and Foxn1Δ/Δ (n = 6); p>0.05 and E13.5 Foxn1+/Δ (n = 5) and Foxn1Δ/Δ (n = 7); p<0.001. (B) E14.5 Foxn1+/+ (n = 7), Foxn1+/Δ (n = 8); p>0.05, Foxn1Δ/Δ (n = 10); p>0.05, Foxn1Δ/nu (n = 9); p = <0.0001, Foxn1nu/nu (n = 6); p<0.0001. (C–G) CD31+ endothelial cells (green) and PDGFR-β+ neural crest mesenchyme (red) can be detected in the thymic capsule and inside the keratin-positive thymus (blue) in E14.5 (C) Foxn1+/+ (D) Foxn1+/Δ (E) Foxn1Δ/Δ (F) but in the capsule only in Foxn1Δ/nu (G) and Foxn1nu/nu mice. Scale bar, 100 µm; n = 3.
Figure 4.
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.
Figure 5.
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.
Figure 6.
Thymus vascular patterning altered in Foxn1Δ mice.
Immunofluorescence analysis on frozen sagittal sections of Foxn1+/Δ and Foxn1Δ/Δ newborn thymus for CD31+ (red) and PDGFR-β+ (green) cells in (A–C) Foxn1+/Δ and (D–F) Foxn1Δ/Δ mice. (G) Average mean fluorescence intensity for CD31 in Foxn1+/Δ (n = 8) Foxn1Δ/Δ (n = 6) thymus sections. Asterisks denote statistical significance (P<0.05). Scale bar, 50 µm; n = 3.
Figure 7.
Electron Microscopic Analysis of E18.5 thymus vascular defects.
Electron microscopy analysis of (A–B) Foxn1+/Δ thymus show compact arrangement of cells including endothelial cells and pericytes, while the (C–D) Foxn1Δ/Δ thymus display loose arrangement of cells, vacuolated endothelium, and indistinct vessel walls. Endothelial Cell (EC), Pericyte (P), Lumen of Blood Vessel (L), and Red Blood Cell (RBC) n = 3.
Figure 8.
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.