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) W^41/41 (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.