Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium
- Division of Experimental Immunology, Institute of Advanced Medical Sciences, University of Tokushima, Japan
- Institute for Immunology and Immunotherapy, University of Birmingham, United Kingdom
- Thymus Biology Section, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, United States
- Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Japan
- Division of Genome Medicine, Institute of Advanced Medical Sciences, University of Tokushima, Japan
- Sequencing Facility, Frederick National Laboratory for Cancer Research, National Cancer Institute, United States
- Single Cell Analysis Facility, Cancer Research Technology Program, National Cancer Institute, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
CCL21-expressing medullary thymic epithelial cells (mTECs) in postnatal thymus.
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 …
Figure 1—figure supplement 1
Distribution of Ccl21a-expressing cells in postnatal thymus.
A replicate of thymus section analysis for postnatal Ccl21atdTomato/+ mice as in Figure 1E.
Figure 2 with 1 supplement
CCL21-expressing medullary thymic epithelial cells (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 …
Figure 2—figure supplement 1
Distribution of Ccl21a-expressing cells in embryonic thymus.
A replicate of thymus section analysis for E15 Ccl21atdTomato/+ mice as in Figure 2F.
Figure 3
Fate mapping of Ccl21a-expressing cells in the thymus.
(A) Schematic illustration of Cre-dependent Ccl21a-specific EGFP expression in Ccl21a-Cre × CAG-loxP-stop-loxP-EGFP mice. FRT, flippase recognition target site. Neo, neomycin resistance gene. Arrows …
Figure 4 with 3 supplements
Characterization of cTECs that previously transcribed Ccl21a.
(A) Quantitative RT-PCR analysis of Cre and indicated genes (means and standard error of the means [SEMs], n = 3) in cTECs (EpCAM+CD45−UEA1−Ly51+), mTEClow (EpCAM+CD45−UEA1+Ly51− I-Alow), and mTEChig…
Figure 4—figure supplement 1
Purity of isolated EGFP+ and EGFP− cTECs for transcriptomic analyses.
Flow cytometric analysis of indicated cells (n = 3) from Ccl21a-Cre × CAG-loxP-EGFP mice at 2-week-old. Numbers indicate frequency of cells within indicated areas.
Figure 4—figure supplement 2
Transcriptomic profiles of cTECs, medullary thymic epithelial cells (mTECs), EGFP+ cTECs, and EGFP− cTECs.
(A) MA plot of 21,155 genes detected in RNA-sequencing analysis of cTECs and mTECs isolated from B6 mice (Ohigashi et al., 2019). Detected genes are plotted as log2 average total counts versus log2 …
Figure 4—figure supplement 3
Distribution of EGFP+ cells in the thymus.
(A) A replicate of thymus section analysis for postnatal Ccl21a-Cre × CAG-loxP- EGFP mice as in Figure 4D. (B) Distribution of Ly51 in the thymus. Left panel shows representative fluorescence …
Figure 5
Ccl21a-expressing medullary thymic epithelial cells (mTECs) during early thymus organogenesis.
(A) Immunofluorescence analysis of thymus section from Ccl21a-Cre × CAG-loxP-EGFP E11 embryos. EGFP (green) and Foxn1 (red) were analyzed as indicated. Dashed lines show the outline of the thymus …
Figure 6 with 1 supplement
CCL21-expressing medullary thymic epithelial cells (mTECs) are distinct from RANK-expressing mTECs.
(A) Immunofluorescence analysis of tdTomato (red) and Venus (green) signals in thymus sections from Ccl21atdTomato/+Tnfrsf11a(RANK)Venus mice at indicated embryonic age. White lines indicate the …
Figure 6—figure supplement 1
Purity of isolated TECs for transcriptomic analysis.
Shown are flow cytometric profiles of indicated cells from Ccl21atdTomato Tnfrsf11a(RANK)Venus E17 mice. Numbers indicate frequency of cells within indicated areas.
Figure 7
CCL21-expressing medullary thymic epithelial cells (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 (Ccl21atdTomato/tdTomato) CCL21-deficient mice at …
Figure 8 with 2 supplements
Developmental potential of CCL21-expressing medullary thymic epithelial cells (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+ …
Figure 8—figure supplement 1
Purity of isolated TECs for reaggregation with RelB-KO thymus stroma.
Shown are flow cytometric profiles of indicated cells from Ccl21atdTomato/+ E17 mice. Numbers indicate frequency of cells within indicated areas.
Figure 8—figure supplement 2
Developmental potential of postnatal CCL21-expressing medullary thymic epithelial cells (mTECs).
(A, B) 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+ …
Figure 9
CCL21-expressing medullary thymic epithelial cells (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 …
