Fig 2: The developmental path from arterial ECs via HSC-primed HECs to HSPCs in the AGM region. a Sorting strategy of CD235a-CD45+CD34+ hematopoietic progenitors in CS 15 dorsal aorta. b Identities of five cell populations in CS 15 dorsal aorta visualized by UMAP. c Violin plots showing the expression of feature genes in each cell cluster. d Heatmap showing the average expressions of top 10 DEGs expressed in HSPC (HSPC1/2/3), Myeloid progenitor and Lymphoid progenitor clusters. e Heatmap showing the average expressions of top 10 DEGs in HSPC1 (GJA5+ HSPC), HSPC2 (Cycling HSPC) and HSPC3 (GFI1B+ HSPC) clusters. f PCA plot of vEC, CXCR4+ aEC, HEC, GJA5+ HSPC, Cycling HSPC and GFI1B+ HSPC. g Dot plots showing the scaled expression level of feature genes in the indicated clusters. Expression of four Notch signaling pathway genes is shown at the bottom. h Trajectory analysis by Monocle 2 combining two aEC sub-clusters (CXCR4+ aEC and HEC) from CS 12/13/14 with three HSPC clusters (GJA5+ HSPC, Cycling HSPC and GFI1B+ HSPC) from CS 15 indicates the developmental path from arterial ECs to HSPCs. Dynamic changes of proportion of clusters are shown on the bottom. i Four distinct gene expression patterns along the pseudotime axis inferred by Monocle 2. j Expression of representative genes of each pattern along pseudotime axis inferred by Monocle 2

Fig 3: GATA-1 promotes erythroid differentiation under hypoxia. (A) Benzidine staining of K562 cells in the context of GATA-1 overexpression or knockdown after hemin treatment for 96 h. Hemoglobinized cells were stained dark blue/black. Scale bar, 100 µm. Data are expressed as the mean ± SD values from three independent experiments. (B) Reverse transcription-quantitative PCR analysis of ?-globin expression in K562 cells in the context of GATA-1 overexpression or knockdown, following hemin induction. The expression levels of ?-globin were significantly increased in GATA-1-overexpressing vs. NC-transfected K562 cells under hypoxia, while its expression in GATA-1 knockdown K562 cells was lower compared that in the NC group. (C) Fluorescence-activated cell sorter analysis of K562 cells in the context of GATA-1 overexpression or knockdown after hemin treatment for 96 h. Data are expressed as the mean ± SD values from three independent experiments. The analysis of data obtained in FACS showing the percentage of CD235a+ cells is presented in the lower panel. (D) Representative Wright's-Giemsa staining of K562 cells in the context of GATA-1 overexpression or knockdown after hemin treatment for 96 h. Scale bar, 10 µm. *P<0.05, two-tailed Student's t-test, n=3. GATA-1, GATA binding protein 1; NC, negative control.