Abnormal Nbeal2^−/− megakaryocyte development in populations of cultured bone marrow cells. Immunofluorescence images of megakaryocytes cultured to the terminal proplatelet stage. (A-E) Cells present in fixed 5-day cultures (n = 5 mice and 600 cells for both Nbeal2^−/− and WT) were identified by immunofluorescence microscopy as MKs by size (>10 µm diameter), large/lobulated nuclei (light blue), and expression of lineage-specific CD41 (green) and VWF (red). Individual cells were classified by apparent developmental stage as (A) early, <20 µm in diameter (ZY sections show these cells to be spheroidal); (B) mature, >20 µm without projections (ZY sections show flattening with increased size); (C) extending, round cells with membrane projections; (D) spread, large cells with extensive membrane and tubulin (violet) projections but not showing clearly defined proplatelets; and (E) terminal proplatelets emanate from these very large megakaryocytes. (E) A distinctive pattern of nuclear retraction (light blue) and elaboration of nascent platelets defined by extensions of membrane (visualized by CD41, green) and cytoskeletal α-tubulin strands and loops (violet). (F) Higher-magnification views of nascent-free platelets (i,ii) and a proplatelet bud (iii) from the WT cell having the distinctive platelet tubulin cytoskeletal ring and the presence of VWF (red, not shown; E), which is absent in Nbeal2^−/− platelets (Figure 2C). Panels A-C show left to right YZ (bars = 5 µm) and XY confocal midcell sections and extended focus images (bars = 10 µm); panels D,E show extended focus images (D scale bar = 10 µm; E scale bar = 20 µm; F scale bars = 1 µm). (G) Population distributions of mean proportions of cells in each stage showed significant differences between Nbeal2^−/− and WT (*P < .05; **P < .01, 2-tailed t test), with Nbeal2^−/− cultures showing a markedly higher proportion of cells in both the early and spread stages and fewer in the mature, extending, and proplatelet stages. (H) Distribution of Nbeal2^−/− and WT CD41+ MK cells and ploidy (n = 3 per group) after 5-day cultured cells, revealing increased 2N and greatly reduced 8N and >16N Nbeal2^−/− MKs.