Fig 2: A subset of ECs, HEC/EHT, and IAHCs derived from PDGFRß+ precursors(A–D) Immunohistochemistry of E11 PDGFRß-Cre:mTmG AGM stained with NG2 (A and B) and CD31 (C) and at E10 with Runx1 (D).(A and B) Arrowheads: DP cells; stars: GFP+ mesenchymal cells.(C) Dashed lines: presumptive separation between DP, PDGFRß-S, and DN layers; stars: hematopoietic CD31+GFP+ cells.(E–I) Flow cytometry analyses of E10 (n = 7) and E11 (n = 3) PDGFRß-Cre; tdTomato AGMs, percentage of Tomato+/- cells within (E) PDGFRß+ cells, (F) EC (CD45-cKit-CD31+CD41-), (G) HEC/EHT-enriched population (CD45-cKit-CD31+CD41+), (H) IAHC/HSPC (CD31+cKit+), and (I) MP-enriched population (CD45+CD31-) live cells; error bars: SD; *p < 0.05, **p < 0.005, ***p < 0.005, ****p < 0.0005.(J) CFU-Cs on PDGFRß+/- cells sorted from E10 (n = 8) and E11 (n = 6) AGMs (see Table S6). E11 AGM were ckit+; error bars: SD; ***p < 0.005, ****p < 0.0005.(K) CFU-Cs on Tomato+/- cells sorted from PDGFRß-Cre; tdTomato E10 (n = 6) and E11 (n = 4) AGMs (see Table S7); error bars: SD; ***p < 0.005.(L) PCR on E11 unsorted cells from PDGFRß-Cre; tdTomato and +; tdTomato WT littermates heads and from sorted AGM cells to detect Cre recombinase expression (324 bp). Unpaired t tests or Mann-Whitney tests were used for the statistics.

Fig 3: Transcriptomic differences by scRNA-seq between WT and KO EC, HEC/EHT, and IAHCs(A) Heatmap showing the expression of Pecam1, Kit, Ptprc, and Runx1 identifying these clusters.(B and C) Scatterplots with genes from (A) that are downregulated in IAHC and ECs.(D) Genes associated with BMP/TGFß, NOTCH, and WNT pathways in EC, HEC/EHT, and IAHCs.(E and F) Genes from (D) that are downregulated in IAHC and ECs. Red dots: significantly downregulated genes.(G and H) Selected biological processes significantly overrepresented in genes significantly downregulated in the KO IAHC (G) and in the KO ECs (H).

Fig 4: PDGFRß+ MSCs are essential for hematopoiesis(A) Schema of co-cultures AGM MSCs and BM LSKs or AGM HSPC/ECs.(B–D) Images of P3 MSCs; 100µm. Example of flow cytometry plots (C) and histograms (D) showing PDGFRß expression in MSCs; error bars: SD.(E) Images of PDGFRß WT and mutant E11 AGM MSCs co-cultured with BM LSKs; 100µm.(F) Gating showing the percentage of CD45+ cells derived from MSC-LSK co-cultures.(G and H) Percentage of CD45+ cells 7 days post-co-culture (G) and CFU-C numbers obtained from co-cultures (H) (n = 5/6/5/4 MSC lines) (see Table S5); error bars: SD; *p < 0.05.(I) Images of MSCs co-cultured with WT E11 AGM-derived HPSCs and ECs; 100µm.(J) Percentage of CD45+ cells 7 days post-co-culture; error bars: SD.(K) CFU-Cs obtained from co-cultures with E11 AGM MSCs; error bars: SD; *p < 0.05, **p < 0.01.(I–K) n = 3/3/3 MSC lines (see Table S5).(L) Expression of osteogenic genes in WT/KO Rß-niche (see Table S4).(M) Osteogenic assay.(N) Example of alizarin red staining of PDGFRß WT, HET, and KO MSCs.(O) Percentage of osteogenic MSC lines. WT: 6/6; HET: 6/7; and KO: 2/7. *p = 0.015, **p = 0.006, z test for proportions.(P) Numbers of MSCs at day 0 of osteogenic assay (WT/HET/KO = 6/6/7); error bars: SD.