Extended Data Fig. 3: Characterization of PDGFRA+ cell contributions to the developing aorta and LT-HSCs.
(A) Spatial distribution of Pdgfra-nGFP, NESTIN and aSMA expressing cells in a Pdgfra-nGFP E11.5 AGM. (B) (i) Schematic outline of experiments using E11.5 wild-type or Pdgfra-GFP+ AGM to evaluate comparability of PDGFRA protein and Pdgfra-nGFP positivity. (ii) Flow cytometry of E11.5 AGMs harvested from Pdgfra-nGFP embryos. (iii) Comparison of CFU-F potential of cells expressing Pdgfra-nGFP and cells expressing PDGFRA protein in the E11.5 AGM (n = 5). (iv) Long-term growth of Pdgfra-nGFP and PDGFRA protein expressing CFU-Fs (n = 3). (C) (i) Schematic outline of AGM/PSC OP9 co-culture experiments. (ii) Bright-field images of cultures at 96 h. (iii) Flow cytometry analysis showing CD45/CD31 expression in GFP+ cells at 96 h. (D) (i) Flow cytometry of E9.5 Pdgfra-nGFP+ AGM for CD31 and GFP expression. (ii) CD31 and Pdgfra expression levels in single CD45−, CD31+, CD144+ endothelial and single CD45−, CD31−, CD144− control cells sorted from the E9.5 embryo proper excluding head, limb buds, heart and visceral bud44. (E) Flow cytometry of limb buds harvested from E11.5 Pdgfra-creERT2 R26R-eYFP embryos after a single injection of tamoxifen at E9.5. (F) (i) Flow cytometry of E11.5 AGM harvested from Pdgfra-creERT2 R26R-eYFP embryos after a single injection of tamoxifen at E9.5. (ii) eYFP expression in CD45+ CD31+ cells in (i). (G) Flow cytometry of AGMs harvested from E11.5 Pdgfra-creERT2 R26R-eYFP embryos after a single injection of tamoxifen at E9.5. (H) Confocal images of E11.5 Pdgfra-creERT2 R26R-eYFP embryo yolk sac, placenta, umbilical and vitelline vessels after a single injection of tamoxifen at E9.5. (I) Flow cytometry analysis of donor eYFP+ cells (from neonatal bone marrow following cre-activation at E9.5) in various tissues in primary transplants. (J) Flow cytometry analysis of donor eYFP+ cell (from neonatal bone marrow following cre-activation at E9.5) contributions to various blood cell fractions in the bone marrow, peripheral blood, thymus and spleens in recipients 6 months post-transplantation. Colony sizes: micro colonies (<2 mm, 2–24 cells), small colonies (2–4 mm, >25 cells) and large colonies (>4 mm, >100 cells). FSC-A: forward scatter area. A linear mixed model was used to compare the growth curves (B(iv)). Data were derived from biologically independent samples, animals and experiments.
