CCL21-expressing mTECs in postnatal thymus.

(A, B) Immunofluorescence analysis of thymus sections from 5-week-old Ccl21atdTomato/+ mice. tdTomato fluorescence (white) was detected with Keratin 14 (Krt 14, red) (A) or Ly51 (green) (B). White lines indicate capsular outline of the thymus. Blue lines show cortico-medullary junction. Representative data from at least three independent experiments are shown. c, cortex. m, medulla. Scale bar, 200μm.

(C) Flow cytometric profiles of tdTomato fluorescence expression in EpCAM+CD45-UEA1+Ly51- mTECs (left) and EpCAM+CD45-UEA1-Ly51+ cTECs (right) from Ccl21atdTomato/+ mice (black line histograms) and WT mice (gray shaded histograms). Numbers in histograms indicate frequency of cells within indicated area.

(D) Proportions (means and SEMs, n=3) of tdTomato+ cells in EpCAM+CD45-UEA1+Ly51- mTECs, EpCAM+CD45-UEA1-Ly51+ cTECs, and EpCAM- non-TECs analyzed as in C.

(E, F) Immunofluorescence analysis of thymus sections from 5-week-old Ccl21atdTomato/+ mice. tdTomato fluorescence (white) was detected with CCL21 (red) (E), Aire or DCLK1 (magenta) (F). White lines indicate capsular outline of the thymus. Blue lines show cortico-medullary junction. Representative data from at least three independent experiments are shown. c, cortex. m, medulla. Scale bar, 200μm (E) or 10μm (F).

(G) Distribution of tdTomato-expressing cells in the medullary region defined by Krt14 expression. Left panel shows representative tdTomato (red) and Krt14 (white) fluorescence signals in the thymus sections from Ccl21atdTomato/+ mice. Scale bar, 100μm. Right panel shows fluorescence intensity profiles of tdTomato (red line) and Krt14 (gray line) signals within the region of interest defined by dashed rectangles in the left panel. Medullary regions were equally divided into three areas into pericortical areas (PM) and central area (CM).

(H) tdTomato intensity (means and SEMs, n=3) in indicated areas (G and Fig. S1A) were calculated in comparison with total tdTomato intensity within the ROI. n.s., not significant.

CCL21-expressing mTECs in embryonic thymus.

(A) tdTomato fluorescence (white) detected in thymus sections from E15 Ccl21atdTomato/+ embryos. White lines indicate the capsular outline of the thymus. The image in central region identified by the box in upper panel is magnified in bottom panel. Representative data from at least three independent experiments are shown. Scale bar, 200μm.

(B) tdTomato (red) and GFP (green) fluorescence in thymus sections from E13 Ccl21atdTomato/+ x Foxn1-GFP-transgenic embryos (left). Dashed lines show the outlines for the thymus and parathyroid primordia. Central region of the thymus identified by the box in upper panel is magnified in bottom panel. Scale bar, 100μm. Right panel shows a representative flow cytometric profile for tdTomato and GFP in CD45-EpCAM+ epithelial cells. Numbers in the contour plot indicate frequency of cells within indicated area.

(C-E) Immunofluorescence analysis of thymus sections from E15 Ccl21atdTomato/+ embryos. tdTomato fluorescence (white) was detected with Krt14 (red) (C), CCL21 (red) (D), Aire, or DCLK1 (magenta) (E). Blue lines show the cortico-medullary junction. Representative data from at least three independent experiments are shown. c, cortex. m, medulla. Scale bar, 10μm.

(F) Distribution of tdTomato-expressing cells in the medullary region defined by Krt14 expression. Left panel shows representative tdTomato (red) and Krt14 (white) fluorescence signals in the thymus sections from Ccl21atdTomato/+ E15 embryos. Scale bar, 100μm. Right panel shows fluorescence intensity profiles of tdTomato (red line) and Krt14 (gray line) signals within the region of interest defined by dashed rectangles in the left panel. The medullary regions were equally divided into three areas into pericortical areas (PM) and central area (CM).

(G) tdTomato intensity (means and SEMs, n=3) in indicated areas (F and Fig. S1B) were calculated in comparison with total tdTomato intensity within the ROI. n.s., not significant.

Fate mapping of Ccl21a-expressing cells in the thymus.

(A) Schematic illustration of Cre-dependent Ccl21a-specific EGFP expression in Ccl21a-Cre x CAG-loxP-stop-loxP-EGFP mice. FRT, flippase recognition target site. Neo, neomycin resistance gene. Arrows indicate PCR primers for allele genotyping.

(B, C) Immunofluorescence analysis of thymus sections from 3-week-old Ccl21a-Cre x CAG-loxP-EGFP mice. EGFP fluorescence (green) was detected with Krt14 (red) (B), Aire, or DCLK1 (magenta) (C). White lines indicate the capsular outline of the thymus. Blue lines show the cortico-medullary junction. Representative data from at least three independent experiments are shown. c, cortex. m, medulla. Scale bar, 200μm (B) or 10μm (C).

(D) Flow cytometric analysis of liberase-digested thymus cells isolated from Ccl21a-Cre x CAG-loxP-EGFP mice at 3-week-old. Histograms show EGFP fluorescence expression in EpCAM+CD45- cells (left), EpCAM-CD45+ cells (middle), and EpCAM-CD45- cells (right) prepared from Ccl21a-Cre x CAG-loxP-EGFP mice (black histograms) and littermate control (gray shaded histograms). Numbers in histograms indicate frequency of cells within indicated area.

(E) Proportions (means and SEMs, n=3) of EGFP+ cells in indicated cell populations analyzed as in D.

(F) Flow cytometric profiles of UEA1 reactivity and Ly51 expression in EpCAM+CD45- TECs (left). Histograms show EGFP fluorescence expression in EpCAM+CD45-UEA1+Ly51- mTECs (middle) and EpCAM+CD45-UEA1-Ly51+ cTECs (right) from Ccl21a-Cre x CAG-loxP-EGFP mice (black histograms) and littermate control (gray shaded histograms). Numbers in the histograms indicate frequency of cells within indicated area.

(G) Proportions (means and SEMs, n=3) of EGFP+ and EGFP- cells in EpCAM+CD45-UEA1+Ly51- mTECs (left) and EpCAM+CD45-UEA1-Ly51+ cTECs (right) analyzed as in F.

(H) Flow cytometric analysis of liberase-digested thymus cells isolated from Ccl21a-Cre x CAG-loxP-EGFP mice at 3 weeks old. Contour plots show Aire (top), DCLK1 (bottom), or isotype control detection with UEA1 reactivity or Ly51 expression in EpCAM+CD45- EGFP+ TECs. Numbers in the plots indicate frequency of cells within the indicated area. Representative data from three independent experiments are shown.

Characterization of cTECs that previously transcribed Ccl21a.

(A) Quantitative RT-PCR analysis of Cre and indicated genes (means and SEMs, n=3) in cTECs (EpCAM+CD45-UEA1-Ly51+), mTEClow (EpCAM+CD45-UEA1+Ly51- I-Alow), and mTEChigh (EpCAM+CD45-UEA1+Ly51- I-Ahigh) isolated from Ccl21aCre/+ mice.

(B) MA plot of 20,230 genes detected in RNA-sequencing analysis of EGFP+ and EGFP- cTECs isolated from Ccl21a-Cre x CAG-loxP-EGFP mice at 2-week-old. Detected genes are plotted as log2 average total counts versus log2 fold changes (EGFP+ cTECs/EGFP- cTECs).

(C) Volcano plot analysis of EGFP+ and EGFP- cTECs. Detected genes are plotted as log2 fold changes (EGFP+ cTECs/EGFP- cTECs) versus −log10 FDR p-values. Fourteen genes (red symbols) are more highly detected (log2 fold change > 1.7, FDR p-value < 0.05) in EGFP+ cTECs than EGFP- cTECs, whereas one gene (blue symbol) is more highly detected (log2 fold change < −1.7, FDR p-value < 0.05) in EGFP- cTECs than EGFP+ cTECs.

(D) Distribution of EGFP-expressing cells in the thymus. Left panel shows representative fluorescence signals for EGFP (green) in the thymus sections from Ccl21a-Cre x CAG-loxP-EGFP mice. The medullary region is defined by UEA1 reactivity (red). White lines indicate the capsular outline of the thymus. Blue lines show the cortico-medullary junction. Scale bar, 100μm. Right panel shows fluorescence intensity profiles of EGFP (green line) and UEA1 (gray line) signals within the region of interest (ROI) defined by dashed rectangles in the left panel. Cortical regions were equally divided into three areas into subcapsular cortex (SC). central cortex, (CC), and perimedullary cortex (PC). M, medulla.

(E) EGFP intensity (means and SEMs, n=3) in indicated areas (C and Fig. S1C) were calculated in comparison with total EGFP intensity within the ROI. n.s., not significant.

Ccl21a-expressing mTECs during early thymus organogenesis.

(A) Immunofluorescence analysis of thymus section from Ccl21a-Cre x CAG-loxP-EGFP E11 embryos. EGFP (green) and Foxn1 (red) were analyzed as indicated. Dashed lines show the outline of the thymus and parathyroid primordia. Image in yellow box in the middle of the thymus is magnified in right panel. Scale bar, 100μm. Representative data from two independent experiments are shown.

(B) Single cell RNA-sequencing analysis of Epcam+Foxn1+ TECs from β5t-Venus knock-in mice at indicated age. Normalized log transcript counts of Ccl21a mRNA (x-axis) and cell numbers (y-axis) are plotted. For E14 TECs, β5t-Venus+ and β5t-Venus- TECs were sorted and analyzed in parallel. Numbers in histograms indicate frequency of cells within indicated area.

(C) Flow cytometric analysis of tdTomato expression in EpCAM+CD45- TECs isolated from Ccl21atdTomato/+ mice at indicated age (black histograms) and WT mice (gray shaded histograms). Bottom plots indicate the proportions (means and SEMs, n=3) of tdTomatonegative (black), tdTomatomedium (blue), and tdTomatohigh (magenta) cells within EpCAM+CD45- TECs as defined in top histograms.

(D) Single cell RNA-sequencing analysis of Epcam+ E12 embryonic pharyngeal epithelial cells. Dots indicate UMAP plot of Ccl21a+ Foxn1+ mTECs (red), Ccl21a+ Gcm2+ parathyroid epithelial cells (green), and other cells (gray).

CCL21-expressing mTECs are distinct from RANK-expressing mTECs.

(A) Immunofluorescence analysis of tdTomato (red) and Venus (green) signals in thymus sections from Ccl21atdTomato/+ RANKVenus mice at indicated embryonic age. White lines indicate the capsular outline of the thymus. Images in boxed regions in upper panels are magnified in bottom panels. Representative data from at least three independent experiments are shown.

(B) Flow cytometric profiles of tdTomato and Venus fluorescence signals in EpCAM+CD45- TECs isolated from Ccl21atdTomato RANKVenus mice at indicated embryonic age.

(C) Principal component analysis of RNA-sequencing data of indicated cell populations isolated from Ccl21atdTomato/+ RANKVenus E17 embryos.

(D) Enrichment analysis of ontology for genes that are differently expressed (RPKM > 1, fold change > 1.5 or < 1.5, FDR p-value < 0.05) between CCL21+RANK- and CCL21-RANK+ TECs. Bars show the adjusted p-values of the top five categories enriched in CCL21+RANK- TECs (top) and CCL21-RANK+ TECs (bottom). Numbers in parentheses indicate the number of categorized genes.

(E) RPKM values of indicated genes detected in RNA-sequencing analysis.

CCL21-expressing mTECs in E17 embryos are functional in thymocyte attraction.

(A) Immunofluorescence analysis of CD4, CD8, Krt14, and tdTomato in thymus sections from heterozygous (Ccl21atdTomato/+) control mice or homozygous (Ccl21a tdTomato/tdTomato) CCL21-deficient mice at E17. E17 thymus sections were stained for CD4, CD8, and Krt14. Representative images for Krt14 (white), tdTomato fluorescence (red), and CD8 (green) expressions are shown. Blue lines indicate cortico-medullary junctions, according to the distribution of Krt14-expressing mTECs. tdTomato-positive cells were localized in the medullary regions. Scale bar, 50μm.

(B, C) Graphs show the means and SEMs (n = 3) of the numbers of CD4+CD8- (B) and CD4-CD8+ (C) thymocytes per unit area (0.01 mm2) in the medullary regions. **, P < 0.01; ***, P < 0.001.

Developmental potential of CCL21-expressing mTECs.

(A-D) Immunofluorescent staining of indicated thymus graft sections. Shown in upper panels are Krt14 expression (white). White lines indicate capsular outline of the thymus. Blue lines show Krt14+ medullary region. Scale bar, 500μm. Bottom panels show Krt14 (white) and Aire (magenta) fluorescence signals within yellow boxes in upper panels. Scale bar, 50μm. Grafts represent relB-KO thymic stroma reaggregated with tdTomato+ TECs (A) and tdTomato- TECs (B) as well as fetal thymus lobe from RelB-KO mice (C) and RelB-heterozygous mice (D).

(E) Size of Krt14+ medullary areas (means and SEMs, n=6) in indicated thymus graft sections.

(F) Size of grafted thymus areas (means and SEMs, n=6) in indicated thymus graft sections.

(G) Proportion (means and SEMs, n=6) of Krt14+ medullary areas in the thymus areas in indicated thymus graft sections.

(H) Numbers of Aire+ mTECs per mm2 of Krt14+ medullary areas in indicated thymus graft sections.

(I) Immunofluorescence analysis of tdTomato (red) and Krt14 (white) in indicated thymus graft sections. Scale bar, 50μm.

(J) Numbers of tdTomato+ cells per mm2 of Krt14+ medullary areas in indicated thymus graft sections. All images are representative data from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.

CCL21-expressing mTECs at E17 embryonic and postnatal period.

(A) Immunofluorescence analysis of thymus sections from E17 or 2-week-old Ccl21atdTomato/+ mice. tdTomato fluorescence (red) and Ly51 (green) expression are shown. White lines indicate thymic capsules. Blue lines indicate cortico-medullary junctions. Images in yellow boxes (top panels) are magnified in bottom panels. Representative data from at least three independent experiments are shown. c, cortex. m, medulla. Scale bar, 100μm.

(B) UMAP plots showing the clusters of Ccl21a+ mTECs in single cell RNA-sequencing analysis of thymic epithelial cells isolated from E16 embryonic and 4-week-old postnatal mice. The majority of E16 Ccl21a+ mTECs were Ccl21alow, whereas the majority of postnatal Ccl21a+ mTECs were Ccl21ahigh (Fig. 5B, C).

(C) Flow cytometric profiles of UEA1 reactivity and Ly51 expression in tdTomatonegative EpCAM+CD45- TECs and tdTomatopositive EpCAM+CD45- TECs isolated from Ccl21atdTomato/+ mice at indicated age. Numbers in the contour plots indicate frequency of cells within indicated area. Representative data from three independent experiments are shown.