Figure 2.
α-Actinin-1 depletion in MKs suppresses megakaryopoiesis, as evidenced by the inhibition of MK polyploidization, PPF, and MK migration. (A) The counts of reticulated platelets in the peripheral blood of Actn1f/f and PF4-Actn1−/− mice (n = 20 mice per group). (B) Platelet depletion in Actn1f/f and PF4-Actn1−/− mice was induced by tail vein injection of an anti-CD42b antibody, after which platelet counts were monitored at different time points (n = 9 mice per group). (C) Splenectomies were performed on Actn1f/f and PF4-Actn1−/− mice, and platelet numbers in the peripheral blood were counted at the indicated time points (n = 9 mice per group). (D) The percentage of progenitors among the BM nucleated cells (n = 7 mice per group). (E) Representative cross-sectional images from 4 experiments. Femurs (BM, left panels) and whole murine spleens (right panels) from Actn1f/f and PF4-Actn1−/− mice were H&E stained. The MKs are indicated by yellow arrows. The scale bar is 50 μm. (F) The area of MKs in the BM and spleen cross-sections was measured for Actn1f/f and PF4-Actn1−/− mice (n = 80 platelets per group). (G) Quantification of the number of MKs in BM and spleen cross-sections from Actn1f/f and PF4-Actn1−/− mice (n = 20 fields per group). (H) The percentage of MKs among all nucleated cells in the BM (upper panel) and spleen (lower panel) (n = 14 mice per group). (I) Representative TEM images of MKs in the BM of Actn1f/f and PF4-Actn1−/− mice. Immature MKs (stage 1) have a single large nucleus and no granules; intermediate MKs (stage 2) have a lobulated nucleus and contain immature, platelet-specific granules; and fully mature MKs (stage 3) have a mature demarcation membrane system (DMS) and contain mature α-granules and dense granules. The scale bar is 5 μm. (J) The percentage of each type of MK in the BM of Actn1f/f and PF4-Actn1−/− mice (n = 10 mice per group). (K) Colony-forming unit (CFU)–MK colonies formed from BM Lin− progenitor cells. The number of small (3-20 MKs), intermediate (21-50 MKs), and large (>50 MKs) colonies and the total number of colonies per slide were calculated for 5 independent experiments (n = 5 slides per group). (L) Detection of polyploidy in MKs from Actn1f/f and PF4-Actn1−/− mice by flow cytometry. (M) Bar graph showing the level of nuclear ploidy in Actn1f/f and PF4-Actn1−/− mice examined by flow cytometry (n = 22 mice per group). (N) Representative images from 5 independent experiments of proplatelet formation. MKs were cultured in vitro from the fetal livers of Actn1f/f and PF4-Actn1−/− mice. Scale bar, 40 μm. (O) The number of PPF-bearing MKs was quantified under a light microscope (n = 10 fields per group). (P) Representative images from 5 independent Transwell experiments of the fetal liver–derived MKs from Actn1f/f and PF4-Actn1−/− mice. The scale bar is 3 mm. (Q) The histogram shows the number of migrated MKs in each field (n = 10 fields per group). ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; LSK, Lin−Scal-1+cKit+; MEP, bipotential MK-erythroid progenitor; MKP, MK progenitor; MPP, multipotent progenitor; ns, not significant; PreMegE, erythroid/MK progenitor; PI, propidium iodide; PPF, proplatelet formation; RP, reticulated platelet.

α-Actinin-1 depletion in MKs suppresses megakaryopoiesis, as evidenced by the inhibition of MK polyploidization, PPF, and MK migration. (A) The counts of reticulated platelets in the peripheral blood of Actn1f/f and PF4-Actn1−/− mice (n = 20 mice per group). (B) Platelet depletion in Actn1f/f and PF4-Actn1−/− mice was induced by tail vein injection of an anti-CD42b antibody, after which platelet counts were monitored at different time points (n = 9 mice per group). (C) Splenectomies were performed on Actn1f/f and PF4-Actn1−/− mice, and platelet numbers in the peripheral blood were counted at the indicated time points (n = 9 mice per group). (D) The percentage of progenitors among the BM nucleated cells (n = 7 mice per group). (E) Representative cross-sectional images from 4 experiments. Femurs (BM, left panels) and whole murine spleens (right panels) from Actn1f/f and PF4-Actn1−/− mice were H&E stained. The MKs are indicated by yellow arrows. The scale bar is 50 μm. (F) The area of MKs in the BM and spleen cross-sections was measured for Actn1f/f and PF4-Actn1−/− mice (n = 80 platelets per group). (G) Quantification of the number of MKs in BM and spleen cross-sections from Actn1f/f and PF4-Actn1−/− mice (n = 20 fields per group). (H) The percentage of MKs among all nucleated cells in the BM (upper panel) and spleen (lower panel) (n = 14 mice per group). (I) Representative TEM images of MKs in the BM of Actn1f/f and PF4-Actn1−/− mice. Immature MKs (stage 1) have a single large nucleus and no granules; intermediate MKs (stage 2) have a lobulated nucleus and contain immature, platelet-specific granules; and fully mature MKs (stage 3) have a mature demarcation membrane system (DMS) and contain mature α-granules and dense granules. The scale bar is 5 μm. (J) The percentage of each type of MK in the BM of Actn1f/f and PF4-Actn1−/− mice (n = 10 mice per group). (K) Colony-forming unit (CFU)–MK colonies formed from BM Lin− progenitor cells. The number of small (3-20 MKs), intermediate (21-50 MKs), and large (>50 MKs) colonies and the total number of colonies per slide were calculated for 5 independent experiments (n = 5 slides per group). (L) Detection of polyploidy in MKs from Actn1f/f and PF4-Actn1−/− mice by flow cytometry. (M) Bar graph showing the level of nuclear ploidy in Actn1f/f and PF4-Actn1−/− mice examined by flow cytometry (n = 22 mice per group). (N) Representative images from 5 independent experiments of proplatelet formation. MKs were cultured in vitro from the fetal livers of Actn1f/f and PF4-Actn1−/− mice. Scale bar, 40 μm. (O) The number of PPF-bearing MKs was quantified under a light microscope (n = 10 fields per group). (P) Representative images from 5 independent Transwell experiments of the fetal liver–derived MKs from Actn1f/f and PF4-Actn1−/− mice. The scale bar is 3 mm. (Q) The histogram shows the number of migrated MKs in each field (n = 10 fields per group). ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; LSK, Lin−Scal-1+cKit+; MEP, bipotential MK-erythroid progenitor; MKP, MK progenitor; MPP, multipotent progenitor; ns, not significant; PreMegE, erythroid/MK progenitor; PI, propidium iodide; PPF, proplatelet formation; RP, reticulated platelet.

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