Extended Data Fig. 4: PDGFRA+ expression is required for LT-HSC generation in the E11.5 AGM.
(A) (i) Schematic showing the experimental strategy used to ablate PDGFRA+ cells. (ii) Phenotypic changes in E11.5 wild-type – and PDGFRA+ – cell-ablated whole embryos (arrows indicate the AGM region). Confocal images of control (Wt / iDTR) (left) and Pdgfra-creERT2 / iDTR (right) E11.5 AGMs following tamoxifen induction at E9.5 and diphtheria toxin treatment at E10.5. (iii) Flow cytometry of control (Wt / iDTR) and Pdgfra-creERT2 / iDTR E11.5 AGM (following tamoxifen induction at E9.5 and diphtheria toxin treatment at E10.5) to quantify numbers of blood (CD45), endothelial (CD31), pericyte (PDGFRB) and CFU-Fs (PDGFRA). (iv) CFU-C quantification in E11.5 AGM (n = 14) after tamoxifen-induced PDGFRA+ cell ablation. (v) CFU-Fs in E11.5 AGM (n = 12) after tamoxifen-induced PDGFRA+ cell ablation. (B) (i) Schematic showing the experimental strategy used to generate Pdgfra-nGFP knockout (KO) embryos. (ii) CFU-Cs in Pdgfra knockout E10.5 and E11.5 AGM (n = 11). (iii) Flow cytometry analysis of donor tdTomato+ cells (from Pdgfra knockout and wild-type AGM) in peripheral blood of recipient mice at 6 months post bone marrow transplantation. CFU-F: colony-forming unit–fibroblast; 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. Ao: aortic lumen; CFU-C: colony-forming unit–culture; CFU-E: colony-forming unit–erythroid; BFU-E: burst-forming unit–erythroid; CFU-GM colony-forming unit–granulocyte/macrophage; Mix: CFU-C with indistinct boundary, possibly co-localization of separate CFU-C or an early split from a single CFU-C. Pdgfra KI/KI (null): –/– and Pdgfra KI/ + (heterozygote): +/–. * p < 0.05, ** p < 0.01, *** p < 0.005; two-sided t-tests were used to compare marker positivity (A(iii)), and random effects Poisson regression was used to compare colony counts (A(iv), (v)) and B(ii)). Data were derived from biologically independent samples, animals and experiments. Data represent mean ± S.D. The precise p values are listed in the source data.
