Fig 2: Functional HSCs Are Localized in 1- to 2-Cell IAHC Cells(A–D) Sections through dorsal aorta of E11 (43 somite pairs, sp) Gata2Venus (green in A and B) and Ly6aGFP (green in C) and of E10.5 (36sp) Csf1rGFP (MacGreen; green in D) embryos. Nuclei are indicated in blue (DAPI), endothelial and IAHC cells are indicated in red (CD34) and CD27+ cells are indicated in white. v, ventral, d, dorsal.(A) Arrowhead indicates small cluster containing two G2+CD27+ cells attached to one G2+CD27- cell. Arrow indicates a single rounded/bulging G2+CD27+ cell.(B) In (Bi), ventral cluster with two G2medCD27+ cells (one bulging out of endothelium). (Bii) Ventral two-cell cluster (G2medCD27+ and G2loCD27- cells). (Biii) Single rounded bulging G2+CD27+ cell on dorsal side of aorta.(C) In (Ci), single Ly6aGFP+CD27+ cell (arrowhead) at apex of bulging Ly6aGFP+ cell within ventral endothelium. A neighboring GFP+ cell in the ventral endothelium is CD27- (arrow). (Cii) Two round CD27+ cells (one is Ly6aGFPlo, indicated by arrowhead) adjacent to each other and attached to interspersed Ly6aGFP+ endothelium. (Ciii) Three CD27+ cells (indicated by arrowheads; one is Ly6aGFP+) in very close association with ventral endothelium. White dashed boxes indicate magnified areas.(D) One CD27+CD34+ cell on dorsal endothelial wall closely associated with a macrophage. White dashed box indicates area for which MacGreen and CD27 expression is shown.Scale bars, 20 µm.

Fig 3: Impairment in T?d17 commitment in KI-PRS mice occurs in immature CD24posCD73neg progenitors. Thymuses from 8-week old KI-PRS and wild-type (WT) littermates were harvested and processed for flow cytometry analysis of T?d cell subsets. (A) Schematic representation of the developmental stages of T?d progenitors based on CD24 and CD73 expression. (B) Representative dot plots show CD24 against CD73 expression among T?d thymocytes (gated as CD3posTCR?dpos). Graph represents the absolute cell number in the quadrant regions shown in 2a (*p-value= 0.0286). (C) representative dot plots show ROR?t expression in the different stages of maturation. Graph represents the frequency of ROR?t cells in quadrant a,b,c,d (*p-value= 0.0286, #p-value= 0.0286) (D) Representative dot plots show CD44 and CD27 expression among immature T?d progenitors (gated as CD3posTCR?dposCD24posCD73neg). Graph represents the frequency of cells in the indicated gates (*p-value= 0.0286, #p-value= 0.0286). (E) Top, representative dot plots show CD44 against ROR?t expression among immature T?d progenitors (gated as CD3posTCR?dposCD24posCD73neg). Graphs represent the frequency and absolute cell number of ROR?t-expressing cells among immature T?d progenitors. Bottom, representative dot plots show CD45RB expression among ROR?t-expressing immature progenitors (gated as CD3posTCR?dposCD24posCD73negROR?tpos). Graphs represent the frequency and absolute cell number of CD45RB-expressing cells among ROR?t-expressing immature progenitors (*p-value= 0.0286, #p-value= 0.0286). All graphs represent mean ± sd of n=4 mice of each genotype. Statistical analysis was performed using Mann-Whitney t-test. ns, non significant. Data are representative of 2 independent experiments.

Fig 4: All Functional AGM HSCs Are within the HC1 CD27med Fraction(A) Violin plots of Tnfrsf7 (CD27) expression in the 5 major transcriptomic clusters. EC, endothelial-like cluster; HC1, hematopoietic cluster 1; HC2, hematopoietic cluster 2; UC1, unknown cluster 1; UC2, unknown cluster 2.(B) Enrichment sorting parameters for CD27+ HC1 cell isolation. Side scatter (SSC-A), CD31, cKit, Gata2 (G2), and CD27 sorting gates and percentage (±SEM) of cells. CD27+ cells (%) are indicated within a wide (+W) and a narrow (+N) mean fluorescent intensity (MFI) gate.(C) Analysis of HSC function within CD27 fractions (CD27+W, CD27+N, and CD27-/lo) of HC1 cells by in vivo transplantation. Open squares denote CD27+W HC1 cell recipients (n = 2) of 442 cells (2.5ee) and 479 cells (3.3 ee) from 15 and 9 (42–50 somite pairs, sp) embryos, respectively. Closed squares (n = 4; 42–49 sp; embryos/experiment: 25, 11, 22, 28) denote recipients injected with CD27+N HC1 cells (238–957; 3.7–9.8 ee), EC cells (899–2,333; 3.9–8.6 ee), CD27-/lo HC1 cells (1,042–4,325; 4.1-9.8 ee), and UC1+2+HC2 cells (1,361–3,741; 4.2–9.9 ee). Percentage of donor cell chimerism in peripheral blood of recipients at 4 months post-injection. Black line indicates mean.(D) Single-cell RNA-seq of 119 CD31hiSSClocKithiGata2medCD27+N sorted cells (all passed quality control) from 6 embryos (41–47 sp). These data combined with the previous dataset (gray) resulted in an extended dataset SPRING plot of 1,087 cells in total. Overlay of CD27+N cells (red) showing localization to one HC1 subcluster (circled).(E) Single CD27+N HC1 cells from E11 G2V IAHCs (50 embryos; 42–49 sp) deposited into methylcellulose and colony-forming unit-culture (CFU-C) counts at 8–10 days (138 total CFU-Cs). Colony type (individual dot) plotted against log10-normalized mean fluorescence intensities (MFIs) for (Ei) CD27, (Eii) G2, (Eiii) cKit, and (Eiv) CD31 expression. GEMM, granulocyte-erythroid-macrophage-megakaryocyte; GM, granulocyte-macrophage. Red lines indicate mean ± SEM.(F) Representative FACS plot showing newly defined CD27med MFI range (red dotted line) within the CD27+N fraction. Percentage ±SEM.(G) Summary of enrichment strategy. The HC1 fraction containing all HSCs is 10.5-fold enriched compared to previous sorting strategies (Eich et al., 2018). Further enrichments are based on gating of specific CD27+N and CD27med fractions to give an additional 6.5-fold enrichment. After iterative analyses and refinement of sorting gates, a total combined HSC enrichment of 68.3-fold is achieved.

Fig 5: Mutations in the polyproline sequence of CD3e impair T?d17 commitment in the thymus. Thymuses from 8-week old KI-PRS and wild-type (WT) littermates were harvested and processed for flow cytometry analysis of T?d cell subsets. (A) Representative dot plots show CD3 and TCR?d expression in total thymocytes. Graphs represent the frequency (left graph) and absolute cell number (right graph) of total T?d cells gated as CD3posTCR?dpos. (B) Representative dot plots of CD44 and CD27 expression among T?d cells. Graphs represent the frequency (left, **p-value= 0.0047) and absolute cell number (right, ##p-value= 0.0070) of T?d17 cells, gated as CD3posTCR?dposCD44hiCD27neg. (C) Representative histograms show the expression of the transcription factor ROR?t among T?dposCD44hiCD27neg cells (T?d17) and T?dposCD44int/lowCD27pos cell (uncommitted T?d). Graph represents the mean of fluorescence intensity (MFI) of ROR?t among the indicated populations. Analysis of IL-17A production. Total thymocyte suspensions were stimulated with PDBu/Io for 6h in presence of Golgi-Plug, and IL-17A production was determined by flow cytometry. Graph represents the frequency of IL-17A-producers among CD44hiCD27neg and CD44int/lowCD27pos T?d cells. (D) Frequency of ?dT cells IL17a producers. (E) Representative dot plots show V?2 expression among T?d17 cells (gated as TCR?dposCD44hiCD27neg). Graphs represent the frequency (left, **p-value= 0.0019) and absolute cell number (right, ##p-value= 0.0019) of V?2pos and V?2neg cells among T?d17 cells. (F) Representative dot plots show V?2 expression among uncommitted T?d cells (gated as TCR?dposCD44int/lowCD27pos). Graphs represent the frequency (left, ***p-value= 0.0002) and absolute cell number (right) of V?2pos and V?2neg cells among uncommitted T?d cells. The inset numbers in representative dot plots represent the percentage of cells within the indicated gate. All graphs represent mean ± sd of n=7-8 mice of each genotype. Statistical analysis was performed using Mann-Whitney t-test. ns, non significant. Data are representative of 2 independent experiments.