MPL is dispensable for formation of DPFCs and MKs in vivo and for the first thrombopoietic phase, but is critical for the rapid expansion of platelet mass during late fetal life. (A) (i) Representative flow cytometry plot demonstrating the expression of MPL at high level by CD45⁻CD41^highCD42D⁺ DPFCs (red) and at low level by CD45⁺CD41^low HPCs (blue) in E10.5 YS cell suspensions. n = 3. Clear curve, isotype control for anti-MPL staining in the HPC population. (ii) qRT-PCR detection of Thpo expression in E8.5 to E10.5 tissues. Expression values are expressed using the ΔΔCt method relative to adult liver expression. Water and RNA from the HPC7 hematopoietic cell line were used as negative controls; RNA from adult liver was used as a positive control. n = 3. Bars, SEM. *RNA was extracted from whole embryo proper at E8.5 and E9.5; at E10.5, RNA was extracted from dissected livers. (B) Quantification of CD41^highCD42D⁺ cells after culture of E10.5 YS DPFCs and HPC for 72 hours in vitro in the presence or absence of exogenous THPO (100 ng/mL). Data are cumulative of 3 independent experiments. ns, not significant. *P < .05. (C) Representative 3-dimensional projection of confocal z-stacks from a Mpl^−/− E10.5 YS revealing the presence of both CD41^highCD42D⁺ DPFCs (arrow) and platelets in vivo (arrow head). n = 15. Bar, 15 μm. (D-E) Quantification of (D) E10.5 YS DPFCs and (E) circulating E10.5 peripheral blood (PB) platelets in wild-type (n = 16) and Mpl^−/− (n = 15) embryos. ns, not significant. Dashed line represents the upper limit of maternal platelet contamination to each sample. (F-G) Numbers of (F) CD41^highCD42D⁺ MKs and (G) circulating peripheral blood platelets in wild-type and Mpl^−/− embryos at E14.5 and E16.5 reveal that Mpl^−/− fetuses are capable of generating normal numbers of MKs, yet exhibit an inability to generate normal numbers of circulating platelets. ns, not significant. ****P < .00001. n = 7 to 16 per genotype. (H) Representative histograms of CD41^highCD42D⁺ liver MK DNA ploidy analysis from (i) E16.5 wild-type and Mpl^−/− and (ii) quantitation of MK ploidy class frequencies at both E14.5 and E16.5 reveal that, although wild-type MKs reach up to 32n in vivo, Mpl^−/− MKs exhibit a pronounced block at the 8n state.