Figure 3.
Figure 3. Thymic architecture of DKO recipient mice remains altered after WT progenitor entry. (A–G) Analysis of WT and DKO thymi without prior transfer. (A) Immunofluorescence microscopy of thymus cryosections from WT and DKO mice. Sections were stained with anti-cytokeratin 5 (Ker5, red) and anti-CD25 (green) antibodies. Arrowhead indicates accumulation of CD25hi cells (DN2 and DN3 thymocytes) at the SCZ of WT thymus. Representative sections of three to four thymi/genotype from two independent experiments are shown. (B) Quantification of medullas per section (sec.), their total area, perimeter and cortex to medulla ratios on cryosections of WT and DKO mice. Each dot represents one section of an entire thymus per mouse analyzed in two independent experiments. Statistical analysis was performed using unpaired Student’s t test, where ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. (C) Light-sheet fluorescence microscopy (LSFM) of WT and DKO thymi. Medullas were labeled with UEA-1 (red). Representative images of virtual sections of five mice/genotype analyzed in two independent experiments are depicted. (D) Quantification of 3D areas of medullary regions (left) visualized by LSFM and their contribution within total thymic surface area (right). (E) Frequencies of mTECs (CD45−EpCAM+UEA-1+), cTECs (CD45−EpCAM+Ly51+), endothelial cells (CD45−CD31+), and fibroblasts (CD45−EpCAM−CD31−CD140a+) shown as percentage of nonhematopoietic cells (CD45−). Pooled data from three independent experiments are depicted. n = 3–6/genotype. Data are represented as mean + SEM. (F) Gene expression analysis of sorted nonhematopoietic cells of WT and DKO mice by qRT-PCR. mTECs were analyzed for expression of Ccl19 and Ccl21a, cTECs were analyzed for expression of Ccl25, thymic endothelial cells were analyzed for expression of P-selectin (Selp), and fibroblasts were analyzed for expression of Flt3l. Experiments were performed with RNA samples isolated from 5–7 pooled thymi for each genotype. Data from one representative experiment are depicted. (G) Quantification of distance of CD25+ cells from SCZ. 200 × 1,600 µm areas were defined on two different regions of section (two random sections for two mice per genotype from two independent experiments as shown in A) using ImageJ software. CD25+ cells were counted on 200 × 200 µm squares. Data are represented as mean ± SEM. (H) Immunofluorescence microscopy of thymus cryosections from WT and DKO recipient mice 21 d after transfer of eGFP-tagged BM precursors. Sections were stained with anti-cytokeratin 18 Ab (Ker18, blue) and anti-cytokeratin 5 (Ker5, red) to visualize thymic cortex and medulla. Arrowheads indicate single eGFP-positive cells (green). Representative sections from three mice for each genotype in one experiment are depicted. Mice used for experiments depicted in Fig. 3 were 4–6 wk old. Donor mice in H were 11 wk old.

Thymic architecture of DKO recipient mice remains altered after WT progenitor entry. (A–G) Analysis of WT and DKO thymi without prior transfer. (A) Immunofluorescence microscopy of thymus cryosections from WT and DKO mice. Sections were stained with anti-cytokeratin 5 (Ker5, red) and anti-CD25 (green) antibodies. Arrowhead indicates accumulation of CD25hi cells (DN2 and DN3 thymocytes) at the SCZ of WT thymus. Representative sections of three to four thymi/genotype from two independent experiments are shown. (B) Quantification of medullas per section (sec.), their total area, perimeter and cortex to medulla ratios on cryosections of WT and DKO mice. Each dot represents one section of an entire thymus per mouse analyzed in two independent experiments. Statistical analysis was performed using unpaired Student’s t test, where ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. (C) Light-sheet fluorescence microscopy (LSFM) of WT and DKO thymi. Medullas were labeled with UEA-1 (red). Representative images of virtual sections of five mice/genotype analyzed in two independent experiments are depicted. (D) Quantification of 3D areas of medullary regions (left) visualized by LSFM and their contribution within total thymic surface area (right). (E) Frequencies of mTECs (CD45−EpCAM+UEA-1+), cTECs (CD45−EpCAM+Ly51+), endothelial cells (CD45−CD31+), and fibroblasts (CD45−EpCAM−CD31−CD140a+) shown as percentage of nonhematopoietic cells (CD45−). Pooled data from three independent experiments are depicted. n = 3–6/genotype. Data are represented as mean + SEM. (F) Gene expression analysis of sorted nonhematopoietic cells of WT and DKO mice by qRT-PCR. mTECs were analyzed for expression of Ccl19 and Ccl21a, cTECs were analyzed for expression of Ccl25, thymic endothelial cells were analyzed for expression of P-selectin (Selp), and fibroblasts were analyzed for expression of Flt3l. Experiments were performed with RNA samples isolated from 5–7 pooled thymi for each genotype. Data from one representative experiment are depicted. (G) Quantification of distance of CD25+ cells from SCZ. 200 × 1,600 µm areas were defined on two different regions of section (two random sections for two mice per genotype from two independent experiments as shown in A) using ImageJ software. CD25+ cells were counted on 200 × 200 µm squares. Data are represented as mean ± SEM. (H) Immunofluorescence microscopy of thymus cryosections from WT and DKO recipient mice 21 d after transfer of eGFP-tagged BM precursors. Sections were stained with anti-cytokeratin 18 Ab (Ker18, blue) and anti-cytokeratin 5 (Ker5, red) to visualize thymic cortex and medulla. Arrowheads indicate single eGFP-positive cells (green). Representative sections from three mice for each genotype in one experiment are depicted. Mice used for experiments depicted in Fig. 3 were 4–6 wk old. Donor mice in H were 11 wk old.

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