Abstract
The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell–restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.
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Acknowledgements
We thank N. Sakaguchi (Kumamoto University) for the Rag1-GFP mouse line; M. Reth (Max Planck Institute of Immunobiology) for Cd79atm1(cre)Reth mice; S. Srinivas (University of Oxford) for R26-stop-eYFP mice; T. Rabbitts (University of Oxford) for Rag1-Cre mice; S. Cory (The Walter & Eliza Hall Institute of Medical Research) for vavP-Mcl1–trangenic mice; N. Iscove (Ontario Cancer Institute, University Health Network) for W41 mice; A. Cumano (Institut Pasteur) for OP9 and OP9-DL1 stromal cells; Biomedical Services at Oxford University for animal support; B. Wu for technical assistance; P. Sopp and S-A. Clark (WIMM FACS facility, supported by the MRC HIU; MRC MHU (MC_UU_12009); NIHR Oxford BRC and the John Fell Fund (131/030 and 101/517), the EPA fund (CF182 and CF170) and by WIMM Strategic Alliance awards G0902418 and MC_UU_12025); C. Lagerholm and D. Waithe (Wolfson Imaging Centre Oxford, supported by the MRC via the WIMM Strategic Alliance (G0902418), the MHU (MC_UU_12009), the HIU (MC_UU_12010), the Wolfson Foundation (18272), MRC/BBSRC/EPSRC (MR/K015777X/1), and MIC –Nanoscopy Oxford (NanO): Novel Super-resolution Imaging Applied to Biomedical Sciences, Micron (107457/Z/15Z)) and the WIMM Strategic Alliance (G0902418 and MC_UU_12025)). Supported by the Medical Research Council UK (G0801073 and MC_UU_12009/5 to S.E.W.J.), the Swedish Research Council (S.E.W.J.), the Knut och Alice Wallenberg Foundation (WIRM; S.E.W.J.), the Tobias Foundation (S.E.W.J.), StratRegen, KI (S.E.W.J.), EU-FP7 EuroSyStem Integrated projects (S.E.W.J., C.C.B.), the Swedish Research Council Linnaeus (Hemato-Linné grant to S.E.W.J.), the MRC (Molecular Haematology Unit core award to M.F.T.R.d.B.), EMBO-LTF (T.C.L.), Kay Kendall Leukaemia Fund (T.C.L.) and Leukaemia and Lymphoma Research (C.C.B. and A.J.M.).
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Contributions
T.C.L., S.L. and S.E.W.J. conceptualized and designed the overall research, analyzed the data and wrote the manuscript, which was subsequently reviewed and approved by all authors; T.C.L. did fate-mapping analyses; S.L. and I.G. established and performed embryonic staging experiments; T.C.L., S.L., H.B., M.F.T.R.d.B. and I.G. performed dissections; T.C.L, T.M., H.B. and E.A. did imaging analyses; T.C.L., S.T., C.B. and I.C.M. analyzed RNA-sequencing data; P.S.W. and A.G. processed RNA samples for RNA sequencing; T.C.L., S.L. and P.S.W. performed in vivo transplantation; T.C.L., S.L., M.L. and T.B.-J. carried out in vitro lineage-potential analyses; T.C.L., H.V., A.J.M. and D.A. did single-cell and quantitative PCR analysis; T.C.L., S.L. and J.C. carried out flow-cytometry experiments; T.C.L., S.L., H.B. and A.J.M. performed morphological analysis; and R.P.,F.G., C.N., R.S., M.F.T.R.d.B., C.C.B. and I.G. contributed advice and input on experimental design and data analysis.
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Integrated supplementary information
Supplementary Figure 1 Staging of fetal thymus at E11.
Related to main Figure 1. (a) Staging criteria for E11.0, 11.25, 11.5 and 11.75 embryos with the corresponding somite (S) pairs indicated. Stage criteria (in red) include morphological assessment of branchial arches (1st and 3rd row), nasal processes (2nd and 3rd row) and limb bud (4th row) development. Abbreviations: BA: branchial arches, numbered; M: Maxillary; m: mandibulary. (b) Tail somite (TS) counting to stage thymus development. Somite development exhibits a faithful correlation with organ development in the embryo, however some somites especially trunk somites have already initiated their differentiation at E11, resulting in ambiguous and inconsistent somite counts. By correlating somites in the tail, which are still visible at E11, with morphology assessment of branchial arches, nasal processes and limb bud development, TS counts representing the corresponding E11 developmental stages were documented (see Fig. 1a). TS counting should only be undertaken in embryos after E11, when the cloaca (*) is protruding, as similar TS number may be obtained from the base of the limb bud at E10.25-10.75 when the cloaca is not yet protruding. (c) At E11.5 the 3rd pouch derived primordia constituted by the thymus (light red) and parathyroid (dark red) rudiments are attached to the pharyngeal endoderm and surface ectoderm. Separation between the thymus and parathyroid primordia starts around E12.0-E12.5. Scheme adapted from27. (d) Sagittal sections were performed to reveal the fetal thymic lobes which are located in the third branchial arches (arrows) and can be visualized by cytokeratin staining (CK, green; DAPI, blue). I, II and III indicate the 1st, 2nd and 3rd branchial arches, respectively. (e) At E11.5 the cytokeratin (CK, red left panel, green right panel) positive fetal thymus is not vascularized but surrounded by blood vessels as confirmed by Vwf eGFP (green left panel, blue right panel) and VE-Cadherin (VE-Cad, red; right panel) staining. Arrows indicate the location of the thymus rudiment.
Supplementary Figure 2 Initial seeding of the early embryonic thymus by cells expressing Rag1-GFP but not by HSCs.
Related to main Figures 1,2,3. (a) Surrounding blood vessels (VE-Cad+) are typically completely removed in dissected fetal thymus lobes (at E11.5 and E12.5) (VE-Cad, red; CK, green; DAPI, blue). (b) Fetal liver cells showing overlap in staining with anti-Vwf antibody (green, left) and Vwf eGFP (blue, middle), as shown in the merged image (right). (c,d) Representative FACS profiles (c) and summary (d) of repopulation activity of total thymocytes (fetal thymus; FT) from 4-5 pooled E11.5 embryos or total fetal liver (FL) cells from 3 pooled E12.5 embryos (CD45.2), transplanted intrafemorally into each irradiated (600Gy) W41/41 (c-kit deficient; CD45.1) recipient. Analysis was performed 2 weeks post-transplantation, since recipients transplanted with FT (but not FL) cells at this time became severely ill and had to be terminated. Data are means (s.d.) from 5 recipient mice receiving FT and 4 recipient mice receiving FL cells. (e,f) Bone marrow cells (25x106) from primary recipients (from c,d) were re-transplanted into lethally irradiated CD45.1 recipients (together with 2x105 CD45.1 bone marrow support cells) to quantify potential stem cell activity in donor-derived (CD45.2) cells. Representative FACS profiles (e) and summary (f) of long-term FT and FL derived (CD45.2) reconstitution of blood cell lineages, 12 weeks post-transplantation, as percentage of total cells within each lineage. Dotted lines indicate the detection level of reconstitution, based on specificity of the antibodies and the number of events acquired by FACS. Mean (s.d.) data from 4 mice in each group. (g) Rag1-GFP (green) expression in cytokeratin (CK, blue) positive thymus lobes from stained sagittal paraffin sections of TS10-15 Rag1 GFP+ embryos. Scale bars represent 10 µm. (h) Whole mount immunofluorescence analysis of a TS14 embryo. Transverse optical sections showing localization of thymic rudiments (arrows) identified by cytokeratin (CK) staining. nt, neural tube; *, notochord. Scale bars represent 300 µm.(i) Whole mount analysis of thymus colonization by Rag1-GFP (green) T-TIPs. Serial transverse optical sections (2.5µm) of a TS14 thymic lobe marked by cytokeratin (CK, red) staining showing GFP+ cells both lining and inside the thymic rudiment. Scale bars represent 30 µm. (j,k) Gating strategy for FACS purification of (j) CD45+Lin–B220–CD19–c-Kit+Flt3+IL-7Rα+ E11.5 fetal liver LMPPs and (k) E14.5 Lin–CD4–CD8–c-Kit+CD25+ DN2 and E14.5 Lin–CD4–CD8–c-Kit–CD25+ DN3 thymocytes. Numbers represent the mean (from 2 biological replicates) percentage of total cells, for each gated cell population.
Supplementary Figure 3 Combined potential to develop into the T cell lieage and myeloid lineage in thymus at E11.5.
Related to main Figures 4,5. (a) Mean (s.e.m.) frequencies of FACS purified single CD45+Lin–c-Kit+CD25–Flt3+ E12.5 thymic progenitors from WT mice, revealing T cell (n = 48 cells) and myeloid/GM (n = 360 cells) lineage potential in vitro, from 2 and 3 independent experiments, respectively. (b) Representative FACS profile of CD4+CD8+ T cells derived from single cell OP9-DL1 culture of FACS purified CD45+Lin–c-Kit+CD25–Flt3+ cells from E12.5 thymus. (c) Characteristic morphology of myeloid cells derived from single cell cultures of CD45+Lin–c-Kit+CD25–Flt3+ E12.5 thymic progenitors. Representative cells belonging to the monocytic (top row) and granulocytic (bottom row) lineages. Scale bars: 10 µm. (d-f) Three representative clones derived from single CD45+Lin–c-Kit+CD25–Flt3+ E11.5 thymic progenitor cells cultured on OP9-DL1 stroma generating CD4+CD8+ T cells and myeloid cells confirmed by FACS (CD11b+) and/or morphology. Asterisks indicate OP9-DL1 stromal cells. Scale bars: 20 µm. (g,h) Representative clone derived from a single CD45+Lin–c-Kit+CD25–Flt3+Rag1-GFP+ cell from the peripheral blood of E11.5 embryos producing CD4+CD8+ and Thy1.2+CD25+ T cells (g) and myeloid (GM) cells confirmed by FACS (CD11b+NK1.1–; g) and morphology (h). Asterisk in h, indicate OP9-DL1 stromal cells. Scale bars: 10µm. (i) Flow cytometry analysis of eYFP expression in CD11b+F4/80+c-Kit–CD25– monocytes/macrophages, ETPs, DN2s and DN3s from E14.5 Rag1-Cretg/+R26eYFP/+ thymuses. Data are representative of 2 independent experiments with 6 and 7 pooled embryos in each experiment. (j) Flow cytometry analysis of Rag1-GFP expression in CD11b+F4/80+c-Kit–CD25– macrophages, ETPs, DN2s and DN3s from E14.5 Rag1-GFP thymuses. Data are representative of 2 independent experiments with 6 and 8 pooled embryos in each experiment. (k,l) Single cell immunofluorescence analysis of intracellular eYFP localization in sorted eYFP+ and eYFP– CD11b+F4/80+c-Kit–CD25– monocytes/macrophages (k) and eYFP+ CD11b–F4/80–c-Kit+CD25– ETPs (l) from E14.5 Rag1-Cretg/+R26eYFP/+ thymuses. Images representative of 80, 17 and 26 single eYFP+ monocytes/macrophages, eYFP– monocytes/macrophages and eYFP+ ETPs analyzed, respectively. DIC, differential interference contrast. (m,n) Representative FACS profiles and mean (s.d.) frequency (m) of CD45.2 reconstitution of thymocytes in CD45.1 lethally irradiated recipient mice, 3 weeks post-transplantation with a mixture of Rag1-CreTg/+ and R26-stop-eYFPFL/FL (1:1) CD45.2 bone marrow cells. (n) Frequency of eYFP+ cells within CD11b+F4/80+CD4–CD8–c-Kit–CD25– macrophages. Data are means (s.d.) of 4 recipient mice. Values in (m) are mean frequencies of total thymocytes (left plot) and of total CD45.2 thymocytes (right plot). Values in (n) are mean frequencies of total CD45.2 thymus cells (left plot) and of total CD45.2 macrophages (right plot).
Supplementary Figure 4 Lineage potential of thymus at E11.5.
Related to main Figures 4 and 5. (a) Morphology of representative myeloid cells from cultures of E11.5 embryonic thymus. White arrows indicate monocytic cells, and black arrows granulocytic cells. Asterisk indicates OP9/OP9-DL1 stromal cells. Scale bars: 10 µm. (b) Representative acetylcholinesterase stained CFU-Mk colonies from E11.5 fetal liver. Scale bars represent 100 µm. (c) Mean (s.d.) number of DAF+ erythroid colonies generated from E11.5 unfractionated fetal liver cells cultured in semi-solid methylcellulose (n = 4; 50,000 cells/biological replicate). (d,e) Representative flow cytometry analyses of T cell (d) and B cell (e) producing cultures of individual thymic lobes from Rag1-GFP E11.5 embryos. (f) Gating strategy for FACS sorting of Lin–CD45loVE-Cad+c-Kit+ AGM stem/progenitor cells (HSPC). Numbers represent mean frequencies of total cells, from 3 litters analysed in 2 independent experiments. (g) Representative FACS profiles of pooled E12.5 thymuses. Virtually all Rag1-GFP+ cells express CD45 and progenitor markers c-Kit and Flt3 but are negative for lineage markers and CD25. The number in the first plot reflects the percentage of total cells, whereas subsequent plots show frequencies relative to total GFP+ cells. All frequencies are means of 3 litters (6-7 embryos pooled in each litter) from 2 independent experiments.
Supplementary Figure 5 Global gene-expression analysis of T-IPs–ETPs, LMPPs and HSCs at various stages of development.
Related to main Figure 7 Hierarchical clustering by genes (a) or by cell populations (b) indicating the relationship between the different cell populations analyzed, according to their global gene expression profile. In b, hierarchical clustering was performed using the multi-scale bootstrap resampling method. Red values denote Approximately Unbiased (AU) p-values (%), and green values denote Bootstrap Probability (BP) p-values (%).
Supplementary Figure 6 Embryonic expression of Ccr, Pir and Notch-related genes in T-IPs, ETPs and candidate thymus-seeding progenitors.
Related to main Figure 7 and 8. (a) Representative flow cytometry analysis of the expression of CCR6, 7 and 9 in E11.5 T-IPs. Numbers represent mean percentages of data from 3 experiments, with 7-9 pooled embryos per experiment. Grey, isotype control; Red, specific anti-CCR antibody. (b) Heatmap for co-expression (ΔCt values, relative to Hprt) of the highest expressed chemokine receptor genes in single E11.5 CD45+Lin–c-Kit+CD25–Flt3+ T-IPs (n = 85 cells, from 2 biological replicates (each a pool of 5 and 9 embryos). Red indicates high, white intermediate and blue low expression levels. Grey indicates below detection level. Three cells were excluded due to absence of Flt3 amplification. (c) Expression of Pir genes in E11.5 T-IPs (n = 6), as compared to E11.5 AGM HSPCs (n = 3) and E11.5 FL LMPPs (n = 3), as well as intra-thymic E12.5 (n = 3), neonatal (NN; 1 week; n = 3) and Adult (8 weeks; n = 3) ETPs. Expression is presented as mean (s.d.) RPKM. Significant differences between different populations are marked with asterisks (*p<0.05, **p<0.01, ***p<0.001); 0, below detection level. n represents biological replicates. (d) Mean (s.d.) percentage of single E11.5 CD45+Lin–c-Kit+CD25–Flt3+ T-IPs co-expressing Pir, GM and lymphoid affiliated genes while not expressing Mk or E genes. Only cells that amplified Flt3, c-Kit and Hprt (corresponding to 97% of total cells analyzed) were included. (n=85 cells, from 2 biological replicates, each using a pool of 5-9 embryos). (e) Representative flow cytometry analysis PIRA/B expression in E11.5 fetal liver CD45+Lin–B220–CD19–c-Kit+Flt3+IL-7Rα+ LMPPs (n=2), E11.5 CD45+Lin–c-Kit+CD25–Flt3+Rag1-GFP+ cells in circulation (n=3), CD45+Lin–c-Kit+CD25–Flt3+Rag1-GFP+ T-IPs (n=3) and E12.5 CD45+Lin–c-Kit+CD25–Flt3+Rag1-GFP+ ETPs (n=3). Numbers in plots indicate average percentages of all biological replicate (n = 2-3), each replicate from a pool of 5-9 embryos. (f) Myeloid and/or T cell generation from single CD45+Lin–c-Kit+CD25–Flt3+ PIRA/B+ or PIRA/B– cells isolated from circulation of E11.5 embryos and cultured on OP9-DL1 (n=254 cells). Mean (s.d.) frequencies from 3 independent experiments are shown.
Supplementary Figure 7 Colonization of the thymus rudiment by T-IPs in Rbpj-deficient embryos at E11.5.
Related to main Figure 8. (a,b) Whole mount imaging of a RbpjFl/Fl Vav-Cre+/+ Rag1-GFPTg/+ TS11 embryo. (a) Transverse optical section showing localization of thymic rudiments (arrows) identified by cytokeratin (CK, red). nt, neural tube; *, notochord. Scale bars represent 300 µm. (b) Three-dimensional image of the left and right thymic lobes showing the thymus rudiment surrounded by Rag1-GFP+ (green) T-IPs. Note that the thymus rudiment is connected to the third pharyngeal pouch on one side and to the external ectoderm on the other side. Scale bars represent 50 µm. (c-e) Using the “surfaces” function of Imaris software the thymus rudiment was 3D-reconstructed to allow the quantification of the distances to Rag1-GFP+ cells. Image segmentation (d, e) was made based on cytokeratin fluorescence intensity (c) and morphology (see also Supplementary Fig. 1). Scale bars represent 50 µm. (f,g) Serial transverse optical sections (2.5 µm) of the thymic rudiment region from a RbpjFl/Fl Vav-Cre+/+ Rag1-GFPTg/+ (c, Vav-Cre+/+) and a RbpjFl/Fl Vav-CreTg/+ Rag1-GFPTg/+ (d, Vav-CreTg/+) embryo. Images are representative of 8 and 6 thymic lobes from 4 Vav-Cre+/+ and 3 Vav-CreTg/+ embryos, respectively. Scale bars represent 50 µm. (c-g) TPP, thymus-parathyroid primordium; PE, pharyngeal endoderm; SE, surface ectoderm
Supplementary Figure 8 Rbpj-deficient T-IPs at E11.5 show unaffected expression of Notch-related and lineage-affiliated genes.
Related to main Figure 8. (a-d) Single cell gene expression analysis of E11.5 CD45+Lin–c-Kit+CD25–Flt3+ T-IPs from RbpjFl/Fl Vav-CreTg/+ Rag1-GFPTg/+ (n=46 cells from 6 embryos) and RbpjFl/Fl Vav-Cre+/+ Rag1-GFPTg/+ (n=36 cells from 8 embryos) littermate controls from 2 different litters. Only cells that amplified Flt3, c-Kit and Hprt (corresponding to 98% of total cells analyzed) were included. (a) Rbpj (b) Notch receptors and Notch target genes (c) frequency of single cells co-expressing GM and early lymphoid affiliated genes. (d) T cell related genes. (b-d) For additional gene expression analysis of Vav-Cre+ cells only cells in which Rbpj had been deleted were included. For panels (a-d) numbers indicate the frequencies of positive cells. For panels b-d no differences between Cre– and Cre+ cells were statistically significant (p<0.05). (e,f) Impact of Rbpj-deficiency on thymocyte progenitor development during early stages of embryonic development. Representative flow cytometry analysis of thymus rudiments from E13.5 (e) and E14.5 (f) RbpjFl/FlVav-CreTg/+Rag1-GFPTg/+ (Vav-CreTg/+; n=3 and n=4, respectively) and RbpjFl/FlVav-Cre+/+Rag1-GFPTg/+ (Vav-Cre+/+; n=7 and n=11, respectively) littermate control embryos. Numbers represent frequencies of total CD45+ cells. (g) In vitro T cell differentiation of E11.5 RbpjFl/FlVav-CreTg/+ (Vav-CreTg/+) and RbpjFl/FlVav-Cre+/+Rag1-GFPTg/+ (Vav-Cre+/+) T-IPs on OP9-DL1 stroma. Data are representative of 4 Vav-Cre+/+ and 6 Vav-CreTg/+ embryos analysed following 14 days of culture. Numbers represent frequencies of total CD45+ cells.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8 and SupplementaryTables 7–11 (PDF 2634 kb)
Supplementary Table 1
Differentially expressed genes between E11.5 T-IPs and E11.5 HSPCs (XLS 270 kb)
Supplementary Table 2
MetaCore Pathway analysis - Pathways upregulated in E11.5 T-IPs (comparing to E11.5 HSPCs) (XLS 76 kb)
Supplementary Table 3
Common ETP genes (XLS 41 kb)
Supplementary Table 4
Genes specificaly upregulated in E11.5 T-IPs comparing to neonatal (NN) and adult ETPs (XLS 57 kb)
Supplementary Table 5
Differentially expressed genes between E11.5 T-IPs and Neonatal (NN) ETPs (XLS 88 kb)
Supplementary Table 6
Differentially expressed genes between E11.5 T-IPs and Adult (AD) ETPs (XLS 199 kb)
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Luis, T., Luc, S., Mizukami, T. et al. Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors. Nat Immunol 17, 1424–1435 (2016). https://doi.org/10.1038/ni.3576
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DOI: https://doi.org/10.1038/ni.3576
