DPFCs emerge in parallel to the progenitor cell lineage. (A) Representative image of E7.5 and E7.75 YS cultures after 72 hours. Scale bar represents 20 μm. After dissection, YSs were incubated in 0.25% trypsin/EDTA at 37°C for 5 minutes, mechanically dissociated, and then cultured in serum-free medium with 100 ng/mL recombinant mouse THPO (anti-CD41-APC and anti-CD42D-PE antibodies were added to the culture medium). Note that CD41⁺CD42D⁺ cells (arrows) were generated from both stages, but proplatelets (arrowhead) were only from E7.75 cultures (n = 3). Scale bar represents 30 μm. (B) Plots of VECAD, CD41, CD42D, and TER119 expression in pooled E8.5 YSs (n = 3). (C) Distribution of E8.5 CFU-EryP (white), CFU-myeloid/erythroid (gray), and CFU-MK (black) in 1 embryo equivalent of sorted YS cells. Error bars indicate SEM (n = 3). EryP colonies were distinguished from erythroid burst-forming unit (BFU-E; which contain both adult-type red cells and 1 leukocyte lineage) according to morphology. (D) Representative images from cultures of purified E8.5 YS populations cultured in proplatelet medium (with anti-CD41-APC and anti-CD42D-PE) for 72 hours (n = 3). Scale bar represents 30 μm. Although VECAD⁻CD41^high cells generated CD41⁺CD42D⁺ MKs, proplatelet-forming CD41⁺CD42⁺ cells (arrow) were only observed from cultures of VECAD⁻CD41^high cells. (E) Representative plots from Runx1^+/+ (n = 5) and Runx1^Δ/Δ (n = 6) E8.5 YS cells at the 8-sp stage demonstrating that the VECAD⁻CD41^high proplatelet-forming lineage is specified in the absence of RUNX1. Values indicate the numbers of cells detected (mean ± SD). (F) E8.5 Runx1^Δ/Δ YSs were capable of forming CD41⁺CD42D⁺ cells after 72 hours in the proplatelet assay (i); these cells are capable of proplatelet formation in vitro (ii). Values represent the mean ± SEM (n = 3). Scale bar represents 30 μm. (G) Representative plot of CD45 and CD41 expression in E10.5 YS from Runx1^+/+ (n = 6) and Runx1-null (n = 8) embryos demonstrating that in the absence of RUNX1 DPFCs can develop (red gate) while HPCs are completely absent (blue gate). Values indicate the numbers of cells detected (mean ± SD). (H) Representative (n = 6) confocal z-stack of Runx1^Δ/Δ E10.5 YS showing CD41^highCD42D⁺ DPFCs and platelets (arrow) can be produced in vivo despite the complete absence of HPCs. Scale bar represents 10 μm. Inset, optical section of boxed region showing a free platelet (arrow) within the Runx1-null YS. Scale bar represents 5 μm.