Gene targeting of thymus epithelium by means of ES cell-mediated nude mouse blastocyst complementation. (A) Injection of Foxn1^+/+ ES cells into Foxn1^nu/nu blastocysts leads to the development of chimeras in which cells are of either ES cell origin or blastocyst origin. The Foxn1-expressing thymic epithelium is of ES cell origin but not of blastocyst origin because the Foxn1 gene acts in cis but not in trans (27, 28). (B) Screening for thymus formation in chimeric mice. Peripheral blood leukocytes from a WT control mouse (a) and from three chimeras (b–d) were analyzed for the presence of T cells.

Morphological and electron microscope analysis of chimeric thymi. (A–D) Morphological analysis of thymi in Vegf^+/+ (A and C) and Vegf^-/- (B and D) chimeras. Thymi were sectioned at 0.5 μm. In the Vegf^+/+ thymus, blood vessels were embedded in the thymic stroma (A, open arrows). In contrast, vessels in Vegf^-/- thymus (B, open arrows) were septum-associated (B, solid arrows). In addition, the thymus in the Vegf^-/- chimera was segmented into multiple small lobe-like structures (pronounced on the left side in B and apparent in D as an overview). (E and F) Electron microscopic analysis of Vegf^+/+ and Vegf^-/- thymi identified normal microvessels with intact endothelial cell (ec) lining and surrounding pericytes (p) in both types of chimeras (E vs. F). (Scale bar in A, which applies to A and B:50 μm; scale bar in D, which applies to C and D: 300 μm; scale bar in E, which applies to E and F: 6 μm.)

Vegf expression in the developing thymus. (A) Vegf isoforms (Upper) and Hprt (Lower) were amplified by PCR from serial dilutions of cDNAs from total day-15.5 fetal thymus, which includes thymocytes and thymic stroma (lanes 1–3), and from cell sorter-purified CD45⁺Thy1⁺ thymocytes (lanes 4–6). PCR products in ethidium bromide-stained agarose gels are shown. (B) Thymus epithelium expresses Vegf. Thymus epithelium was sorted into cortical (CDR1⁺, lanes 1 and 2) and medullary (A2B5⁺, lanes 3 and 4) TECs. Total thymus (lane 5) and template omission (lane 6) served as positive and negative controls, respectively. Vegf PCR products were positively identified by Southern blotting, using an internal oligonucleotide probe. Thymus-expressed Vegf RNAs include isoforms VEGF₁₂₀ (544-bp PCR product), VEGF₁₆₄ (748-bp PCR product), and VEGF₁₈₈ (820-bp PCR product). Molecular weight (MW) markers are 1,033, 653, and 517 bp.

Loss of Vegf expression in TECs but not in thymic cMes. (A) Expression of the stromal cell markers UEA-1 (green), G8.8 (blue), and Ly51 (red) on thymus sections. UEA-1⁺ cells were located in the medulla (M). Most G8.8⁺ cells were found in the medulla; few were found at the corticomedullary junction (arrow-heads). Ly51⁺ cells were located in the cortex (C). (Scale bar, 120 μm.) (B–E) Flow cytometry on total thymus cell suspensions. CD45^- cells were resolved by UEA-1 vs. Ly51 staining (B). Subpopulations gated according to regions R1, R2, and R3 were analyzed for expression of G8.8 and CD31 (C–E). Medullary TECs were sorted as CD45^-UEA-1⁺Ly51^- G8.8⁺CD31^- cells (C). CD45^-UEA-1^-Ly51⁺ stromal cells (region R2 in B) were separated into cTECs (G8.8⁺CD31^-) and cMes (G8.8^-CD31^-) (D). UEA-1^-Ly51^- stromal cells (R3 in B) lacked expression of G8.8 and CD31 and were designated marker^- (E). (F) Stromal cells were analyzed for expression of lineage hallmark genes by RT-PCR using comparable amounts of cDNAs. All populations expressed the positive control gene Actin (lanes 1–5). Stromal cells were free of thymocyte contamination, as shown by lack of CD3ε expression (lanes 1–4). TEC subsets (mTECs, lane 1; cTECs, lane 3) expressed typical TEC genes: i.e., Foxn1, Aire, Keratins 5, 14, 8, and 18, and thymus growth factors (Scf and Fgf10). In contrast, cMes (lane 2) and the marker^- population (lane 4) lacked TEC gene expression. (G) TEC subsets but not cMes were Vegf-deficient in Vegf^-/- chimeras. Stromal cell subsets defined as in A–F were sorted from WT thymus (lanes 1–3) and from Vegf^-/- chimeras (lanes 4–6) and were analyzed for expression of Vegf (Upper) and for expression of the ES cell marker Neo (Lower). Vegf was expressed in all subsets in the normal thymus; it was absent from both TEC subsets (lanes 4 and 6) but not from cMes (lane 5) in Vegf^-/- chimeras. Expression of Neo RNA in both TEC subsets (lanes 4 and 6) correlates with the ES cell origin of TECs. Lack of Neo RNA in cMes (lane 5) indicates their nude origin. Total WT thymus (lane 7) and template omission (lane 8) served as positive and negative controls.

Angiography of Vegf^+/+ and Vegf^-/- chimeras. (A–F) Vegf^+/+ (A, C, and E) and Vegf^-/- (B, D, and F) chimeras were injected intravenously with the FITC-labeled lectin BS-1. Whole-mount preparations of thymus (A and B), muscle (C and D), and liver (E and F) were examined by fluorescence microscopy (fluorescence in black). Longitudinal blood vessels along muscle fibers (C and D) and fine vascular networks in the liver (E and F) were comparable in Vegf^+/+ and Vegf^-/- chimeras. Morphometric analyses revealed 41 and 40 vessel branches per mm² in muscles from Vegf^+/+ (C) and Vegf^-/- (D) chimeras, respectively, and 183 and 162 branches in the livers from Vegf^+/+ (E) and Vegf^-/- (F) chimeras, respectively. In the thymus in Vegf^+/+ chimeras, blood vessel architecture was characterized by a dense and regular vascular network with frequent branches and loop structures. The Vegf^+/+ chimeric thymus contained 123 vessel branches per mm² of thymus cortex (A). Thymus vasculature in Vegf^-/- chimeras showed reduced vessel density and branching frequencies. The Vegf^-/- chimera thymus contained 39 vessel branches per mm² of thymus cortex (B). In addition, the diameter of vessels in Vegf^-/- chimeras was larger compared with Vegf^+/+ chimeras. (G–J) Angiography is shown at higher magnification for three Vegf^-/- chimeras (G–I) compared with WT thymus (J). Morphometric analyses of Vegf^-/- chimeras revealed 52, 85, and 40 vessel branches per mm² of thymus in G, H, and I, respectively. The control WT thymus harbored 190 branches per mm² of thymus (J).