Identification of diploid platelet-forming cells (DPFCs) in the E10.5 YS. (A) CD45 and CD41 expression in pooled E10.5 YS. Values indicate the numbers (mean ± standard error of the mean [SEM]) of each population per embryo equivalent (n = 15). (B) Distribution of CFUs with MK potential in 1 embryo equivalent of whole or purified E9.5 and E10.5 YSs. Cells were dissociated in 10% collagenase/dispase for 45 minutes at 37°C and then dissociated mechanically. No CD45⁺CD41⁻ cells were present in E9.5 YS. Error bars represent SEM (n = 3). sp, somite pairs. (C) To distinguish embryonic from contaminating maternal platelets in preparations of embryonic peripheral blood, green fluorescent protein (GFP)–expressing male mice were mated with wild-type females, ensuring that all GFP⁺ cells were of embryonic origin. Flow cytometry plots showing the presence of GFP⁺ platelets in the PB of E9.5 (i) and E10.5 (ii) embryos. (iii) Quantification of embryonic platelets in the PB of E9.5–10.5 embryos. Error bars represent standard deviation (SD) (n = 9–21 embryos per stage). (D) (i) Representative image of E10.5 YS CD45⁺CD41^low HPCs cultured in proplatelet medium (including anti-CD41-APC) for 72 hours (n = 5). Red indicates CD41 expression. Scale bar represents 40 μm. (ii) DNA content analysis of HPC-derived CD41^highCD42D⁺ cells demonstrating a conventional MK 2–16n ploidy profile (n = 3). Following antibody staining, cells were fixed in 80% ethanol, permeabilized in TritonX-100, and stained with 4,6-diamidino-2-phenylindole (DAPI). (E) Multidimensional scaling plot of microarray data comparing transcriptional similarity of E10.5 YS CD45⁻CD41^high cells with E10.5 YS lineages (HPC = CD45⁺CD41^low; myeloid = CD45⁺CD41⁻; endothelial = VECAD⁺CD45⁻CD41⁻) and E13.5 fetal liver (FL) lineages (blast = CD45⁺CD11B⁻; myeloid = CD45⁺CD11B⁺; and MK = CD41^highCD42D⁺CD150⁺). Note that myeloid and HPC populations from the YS cluster with their FL counterparts, whereas YS CD45⁻CD41^high cells cluster most strongly with E13.5 FL MKs. (F) DNA content analysis of E10.5 YS CD45⁻CD41^high cells demonstrating that these cells are predominantly 2n in vivo (n = 3). (G) Cumulative frequency plot of in vitro proplatelet-forming capacity of 145 CD45⁻CD41^high pedigrees. Cells were cultured in serum-free medium with 100 ng/mL recombinant mouse thrombopoietin (THPO) (including anti-CD41-APC) and live-imaged at a 3- to 4-minute time resolution. Data from 3 independent experiments are shown. For each experiment, 6000 cells were plated per chamber. The number of microwells imaged was limited by the number of positions that could be imaged within a 3- to 4-minute time period. n = number of pedigrees per experiment. (H) Representative confocal z-stack of E10.5 YS CD45⁻CD41^high cells after 72 hours in serum-free medium with 100 ng/mL recombinant mouse THPO (n = 30). Cultures were fixed in 2% paraformaldehyde (PFA) for 20 minutes, permeabilized with 0.6% TritonX/10% fetal calf serum (FCS), and stained with anti-CD41 and anti-CD42D antibodies (0.3% TritonX/5% FCS); nuclei were stained with DAPI. Note that cells are generating proplatelets while in a diploid state. Scale bar represents 10 μm. (I) Scatter plot of object volumes and relative DNA content of CD41^highCD42D⁺ cells within E10.5 YSs that are in the process of platelet production in vivo. Surface objects for nuclei were generated by manually defining the area of DAPI staining within individual cells using Imaris software (Bitplane); intensity sum values representing the 4n state were generated from mitotic figures from CD41⁻CD42D⁻ cells within the z-stack. Relative DNA content was derived by dividing the intensity sum of CD41^highCD42D⁺ nuclei by that of a mitotic figure within the same z-stack. This strategy allows determination of ploidy of cells within the z-stack that are, or are not, in the process of platelet production. A value of 0.5 represents 2n, and 1.0 represents 4n. Data are derived from 10 individual z-stacks taken from 5 YSs. (J) Representative confocal z-stack of CD41^highCD42D⁺ cells within the vascular endothelial cadherin (VECAD)–expressing vasculature of the E10.5 YS in vivo. Surface objects of DNA (DAPI) content are overlaid and reflect the range of DNA content of CD41^highCD42D⁺ cells in vivo. The diploid state of a proplatelet-forming cell (blue arrowhead) is highlighted. Freshly dissected YSs were fixed in 2% PFA for 20 minutes, permeabilized with 0.6% TritonX/10% FCS for 30 minutes, and stained with anti-CD41, anti-VECAD, and anti-CD42D antibodies (0.3% TritonX/5% FCS); nuclei were stained with DAPI. Colors reflect the intensity sum of DAPI signal, ranging from the 2n (blue) to 4n (red) states. Scale bar represents 10 μm; n = 20.