Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors

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.

Figure 1. Initial seeding of the embryonic thymic anlage

(a) Correlation of the number of tail somites with the timing of embryonic development. Each circle represents an individual embryo. Area between broken lines corresponds to the tail somite stages (10-12) of initial thymus seeding. (b) Typical dissected thymic lobes from E11.5 embryos stained with VE-Cadherin (VE-Cad, red), cytokeratin (CK, green) and DAPI (blue). VE-Cadherin⁺ endothelial cells were completely removed by dissection in most explants (top), but not all (bottom). (c) Frequency of seeded embryonic thymic rudiments, as determined by emergence of T cell potential of dissected thymuses at tail somites 4-9 (n=18), 10-12 (n=17) and 13-16 (n=9), corresponding to E11.0-E11.75 embryos. p values were calculated with two-sided Fisher’s exact test. (d) Representative FACS profile of T cells (CD4⁺CD8⁺ and/or Thy1.2⁺CD25⁺) derived from culture of an individual E11.5 (TS11) thymus on OP9-DL1 stroma. (e) Expression (2^–ΔCt) of Dll4 in PLET1⁺ thymic epithelial cells isolated from E11.5 and E12.5 embryos. Mean (s.d.) expression levels (relative to the average of Actb, Hmbs and Tbp) of 2 biological replicates are shown. **p<0.01.

Figure 2. Hematopoietic stem cells do not seed the embryonic thymus rudiment

(a) Vwf eGFP (green; marking HSCs and endothelium) and cytokeratin (CK, red) staining in thymus and FL sections of TS8-14 Vwf eGFP⁺ embryos. Scale bars: 10µm. (b) Frequency of E11.0-E11.75 embryos with Vwf eGFP⁺ inside and/or adjacent to/lining the thymus rudiment. The number of embryos investigated is shown in brackets. In each case the complete thymuses were sectioned and analyzed. (c-e) Long-term (HSC) reconstituting activity of total thymocytes (fetal thymus; FT) from 4-5 E11.5 or E12.5 Vwf eGFP embryos or total FL (FL) cells from 3 E12.5 Vwf eGFP embryos, transplanted intravenously (i.v.) or intrafemorally (i.f.) into each lethally irradiated recipient. (c,d) FACS profile of typical peripheral blood of a CD45.1 mouse transplanted i.f. with CD45.2⁺ E12.5 total fetal thymocytes (c) or with CD45.2⁺ E12.5 total FL cells (d) 16 weeks earlier. No thymocyte-derived (CD45.2⁺) cells were observed (detection level 0.003%). Left, percentage CD45.2 contribution to total blood cells, Middle and right, distribution between myeloid cells (Gr1⁺CD11b⁺), B cells (CD19⁺) and T cells (CD4/CD8⁺), within CD45.2⁺ cells. (e) Summary of long-term thymocyte and FL reconstitution of blood cell lineages, 15-17 weeks post-transplantation, as percentage of total cells within each lineage. Numbers above graphs indicate frequency of reconstituted mice (see Online Methods). Dotted lines indicate the detection level of reconstitution for each lineage, based on the number of events acquired by FACS. Data from 5 independent experiments.

Figure 3. Initial seeding of the embryonic thymus by Rag1-GFP⁺ progenitors

(a) Representative Rag1-GFP (green) expression in cytokeratin (CK, blue) stained sections of the thymus rudiment in TS8-16 Rag1-GFP⁺ embryos. Scale bars represent 10µm. (also see Supplementary Fig. 2g-i). (b) Frequencies of E11.0-E11.75 embryos with Rag1-GFP⁺ cells exclusively lining (maximum 2 cell diameter distance from thymic epithelium) or lining and inside the thymus rudiment. Embryos with Rag1-GFP⁺ cells inside the thymic rudiment consistently also showed Rag1-GFP⁺ cells outside or lining the thymus. The number of individual embryos investigated shown in brackets. *; 3 complete thymuses were serially sectioned and analyzed, whereas remaining thymuses were partially analyzed (see Online Methods). (c,d) Representative FACS profiles of pooled E11.5 thymuses. Virtually all Rag1-GFP⁺ cells express CD45 and progenitor markers c-Kit, Flt3 and IL-7Rα but are negative for lineage markers and CD25. The vast majority is also CCR9⁺. The number in the first plot in c, reflects the percentage of total cells, whereas subsequent plots show frequencies relative to total GFP⁺ cells. In d, numbers reflect frequencies relative to CD45⁺Rag1-GFP⁺ cells. All frequencies are means of 2 independent experiments, 8 and 9 embryos pooled in each experiment. (e) Analysis of T-cell progenitor development from E11.5 T-IPs on OP9-DL1 stroma. Representative FACS profiles after 7 and 14 days of culture, of cells generated from individual E11.5 thymic lobes isolated at indicated developmental stages. Data correspond to 3 diffferent embryos, representative of a total of 9 analyzed embryos. Numbers in plots indicate percentages of total CD45⁺ cells.

Figure 4. Thymopoiesis-initiating progenitors have combined T-lymphoid and granulocyte-monocyte lineage potentials

(a) Cytokine receptor, T cell and myeloid lineage affiliated gene expression (ΔCt, relative to Hprt) in single E11.5 CD45⁺Rag1-GFP⁺ T-IPs (n=166), E11.5 CD45⁺Lin⁻B220⁻CD19⁻c-Kit⁺Flt3⁺IL-7Rα⁺ FL lympho-myeloid-restricted progenitors (n=77), E14.5 Lin⁻CD4⁻CD8⁻c-Kit⁺CD25⁺ DN2 (n=42) and E14.5 Lin⁻CD4⁻CD8⁻c-Kit⁻CD25⁺ DN3 (n=41), purified from 2-4 biological replicates, each representing 8-16 embryos. Grey, below detection level. Only Hprt amplified cells (98.8%) were included. (b,c) Cloning frequency (b) and lineage distribution (c) of single CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺ E11.5 T-IPs cultured on OP9-DL1 (n=88 cells), from 4 experiments. (d,e) Representative clone from single CD45⁺Lin⁻c-Kit⁺CD25–Flt3⁺ E11.5 T-IP on OP9-DL1 producing CD4⁺CD8⁺ T cells (d) and myeloid cells confirmed by FACS (CD11b⁺CD4⁻CD8⁻NK1.1⁻; d) and morphology (e). Asterisk in e, indicate OP9-DL1. Scale bars: 20µm. (f) E11.5 circulating CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺Rag1-GFP⁺ cells. Mean percentages of total live cells from 4 litters, 7-9 embryos each. (g) Frequency of clones (left) and myeloid and/or T-cell potential (right), from single circulating E11.5 CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺Rag1-GFP⁺ cells on OP9-DL1. Means of 3 independent experiments (n=254 cells). (h,i) Representative clone from single circulating E11.5 CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺Rag1-GFP⁺ cell producing CD4⁺CD8⁺ T-cells (h) and myeloid cells (h-i). * = OP9-DL1 cells. Scale bars: 10µm. (j,k) Rag1-Cre fate mapping of myeloid cells from pooled TS4-TS8 (j, n=3 experiments) and E14.5 (k,n=2 experiments) Rag1-Cre^tg/+R26^eYFP/+ thymuses, showing eYFP expression (mean frequency) on CD11b⁺F4/80⁺c-Kit⁻CD25⁻ myeloid cells in E14.5 but not TS4-TS8 thymuses. (l) Morphology of representative eYFP⁺ and eYFP⁻ CD11b⁺F4/80⁺c-Kit⁻CD25⁻ monocytic cells from E14.5 Rag1-Cre^tg/+R26^eYFP/+ thymuses. Scale bars: 5µm. (m) Frequencies of monocytes/macrophages (M), granulocytes (G), Immature myeloid (IM) and lymphocytes (Ly) determined by morphology of sorted E14.5 eYFP⁻ or eYFP⁺ CD11b⁺F4/80⁺c-Kit⁻ CD25⁻ cells. Mean (s.d.) results from 2 experiments (4-6 pooled embryos; n=152 eYFP⁻ cells and n=199 eYFP⁺ cells).

Figure 5. Embryonic thymopoiesis is initiated by Rag1-GFP⁺ lympho-myeloid restricted progenitors

(a) T cell, B cell, myeloid (granulocyte-macrophage; GM), megakaryocyte (Mk) and erythroid (E) potential of dissected individual thymuses from embryos at TS10-12 (n=7-22 embryos investigated for each lineage) and TS13-16 (n=9-21 embryos). (b-d) B cell (TS8-9 and TS10-11) and T cell (TS10-11) potential of individual thymic lobes following culture on OP9 and OP9-DL co-cultures, respectively. Shown is the percentage of embryos with one (grey) or both (black) thymic lobes exhibiting T cell (CD4/CD8⁺Rag1-GFP⁺ and/or Thy1.2⁺CD25⁺Rag1-GFP⁺) and B cell (B220⁺CD19⁺Rag1-GFP⁺) potential, respectively. The number of embryos investigated is shown in brackets. (d) Typical FACS profiles of cultures with individual thymic lobes showing T cell and B cell potential (see Supplementary Fig. 4d,e for additional profiles). (e) Analysis of pooled E11.5 Mb1-Cre^tg/+R26^eYFP/+ embryos showing lack of eYFP expression in CD45⁺ thymocytes (n=3 and n=5 eYFP⁺ embryos per pool in 2 independent experiments). (f) Single cell gene expression of early B-cell specific genes in E11.5 T-IPs (n=166 cells), E11.5 fetal liver CD45⁺Lin–B220–CD19–c-Kit⁺Flt3⁺IL-7Rα⁺ LMPPs (n=77 cells) and E14.5 Lin⁻ B220⁺CD43⁺CD19⁺CD24⁺AA4.1⁺IL-7Rα⁺c-Kit⁺ ProB-cells (n=41 cells) from 2 biological replicates, shown as mean (s.d.) expression values (relative to Hprt). ***p<0.001 (g) Left; Mean (s.e.m.) frequencies of FACS purified single CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺ E12.5 thymic progenitors from VavP-Mcl1–transgenic mice, possessing T cell and B cell potential (n=96 single cells from 2 experiments). Right; B220⁺CD19⁺ B cells generated from a single E12.5 CD45⁺Lin–c-Kit⁺CD25– Flt3⁺ cell on OP9 stroma cells.

Figure 6. Molecular profiling of E11.5 thymopoiesis-initiating progenitors

(a) Gene-set enrichment analyses of global RNA sequencing data from E11.5 CD45⁺Lin⁻c-Kit⁺CD25⁻ Flt3⁺ T-IPs (n=3) versus E11.5 Lin⁻CD45^loVE-Cad⁺c-Kit⁺ AGM stem/progenitor cells (HSPC) (n=3) for Lymphoid, Pre-MegE and HSC gene sets. NES, normalized enrichment score; FDR, false discovery rate. (b-e) mRNA expression, shown as mean (s.d.) RPKM, of (b) early lymphoid (c) Mk (d) HSC and (e) GM affiliated genes in E11.5 T-IPs (n=3) and AGM HSPCs (n=3). *p<0.05, **p<0.01, ***p<0.001. n represents number of biological replicates. (f) Heatmap for expression (ΔCt values, relative to Hprt) of lineage (HSC, hematopoietic stem cell; Ly, lymphoid; My, myeloid; E, erythroid; Mk, megakaryocytic) affiliated genes in single E11.5 CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺ T-IPs (n=85 cells, from 2 biological replicates, each using a pool of 5 and 9 embryos). Grey indicates not detected. Three cells were excluded due to absence of Flt3 amplification. (g) Mean (s.d.) frequency of single E11.5 CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺ T-IPs co-expressing GM and lymphoid affiliated genes while not expressing Mk or E genes (from f). Only cells that amplified Flt3, c-Kit and Hprt (corresponding to 97% of total cells analyzed) were included. Flt3 and Gata3 were not considered as lymphoid in this analysis due to their concomitant non-lymphoid expression pattern.

Figure 7. E11.5 thymopoiesis-initiating progenitors express a distinct repertoire of chemokine receptors

(a) PCA of normalized global gene-expression of E11.5 Lin⁻CD45^loVE-Cad⁺c-Kit⁺ AGM HSPCs (n=3), E11.5 Lin⁻c-Kit⁺Flt3⁺IL-7Rα⁺ FL LMPPs (n=3), E11.5 CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺ T-IPs (n=6), E12.5 CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺ ETPs (n=3) , E13.5 CD45⁺Lin⁻c-Kit⁺CD25⁺Flt3⁻ DN2s (n=3), and neonatal (NN; 1 week, n=3) and adult (8 weeks, n=3) Lin⁻CD4⁻CD8α⁻c-Kit⁺CD25⁻Flt3⁺ ETPs. Each biological replicate (100 cells) was from a different pool of mice. (b) Heatmap and Venn diagram based on RNA sequencing data, representing genes up-regulated (>4-fold) in CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3⁺ T-IPs and ETPs at indicated developmental stage, relative to HSPCs/HSCs at equivalent stages. (c) Expression of chemokine receptor genes, shown as mean (s.d.) RKPM, in HSPCs/HSCs (n=3), LMPPs (n=3), and T-IPs/ETPs (n=3-6) at different developmental stages (LMPPs and HSPC/HSC were not investigated at E12.5). Significant differences between developmental stages within each population; *p<0.05, **p<0.01, ***p<0.001; 0, below detection level.

Figure 8. Initial colonization of the E11.5 thymus rudiment takes place independently of Notch signaling

(a) GSEA for Notch pathway genes (see Online Methods). NES, normalized enrichment score; FDR, false discovery rate (n=3 biological replicates per population). (b) Notch receptor and target gene expression, shown as mean (s.d.) RPKM (n=3 per population). Significant differences between T-IPs/ETPs at different developmental stages; *p<0.05, **p<0.01, ***p<0.001; 0, below detection level. HSPCs and LMPPs were only analyzed at E11.5. (c,d) Whole mount imaging of E11.5 (TS10-12) thymus colonization by Rag1-GFP⁺ (green) T-IPs in Rbpj^Fl/FlVav-Cre^Tg/+Rag1-GFP^Tg/+ (Vav-Cre^Tg/+) and Rbpj^Fl/FlVav-Cre^+/+Rag1-GFP^Tg/+ (Vav-Cre^+/+) littermate controls. (c) Three-dimensional image (left) and corresponding reconstruction (right) of the thymus rudiment. Rag1-GFP⁺ cells (right) are color coded according to their relative distance to the rudiment surface: Inside (blue) 0 µm; Lining (yellow), >0 and ≤10 µm; Outside (green), >10 and ≤100 µm. (d) Total number of cells inside, lining and outside the thymus rudiment, shown as mean (s.d.) representing 6 and 8 thymic lobes from Rbpj^Fl/FlVav-Cre^Tg/+Rag1-GFP^Tg/+ and Rbpj^Fl/FlVav-Cre^+/+Rag1-GFP^Tg/+, respectively. Thymus rudiment is marked by cytokeratin (CK, red). Scale bars represent 50 µm. (e) Heatmap representation (ΔCt values, relative to Hprt) of Notch-related and lineage-affiliated genes in single E11.5 CD45⁺Lin⁻c-Kit⁺CD25⁻Flt3^-T-IPs from Rbpj^Fl/FlVav-^CreTg/+Rag1-GFP^Tg/+ (n=46 cells from 6 embryos) and Rbpj^Fl/FlVav-Cre^+/+Rag1-GFP^Tg/+ (n=36 cells from 8 embryos) littermate controls from 2 litters. Only cells amplifying Flt3, c-Kit and Hprt (corresponding to 98% of total cells) were included. Grey indicates not detected. (f) Impact of Rbpj-deficiency on thymocyte progenitor development. Mean (s.d.) absolute numbers of ETPs, DN2s and DN3s from E13.5 and E14.5 Rbpj^Fl/FlVav-Cre^Tg/+Rag1-GFP^Tg/+ (Vav-Cre^Tg/+; n=3 and n=4, respectively) and Rbpj^Fl/FlVav-Cre^+/+Rag1-GFP^Tg/+ (Vav-Cre^+/+; n=7 and n=11, respectively) littermate controls. **p<0.01, ***p<0.001.