Deletion of Mef2C in the hematopoietic system reveals a requirement of Mef2C for proper megakaryocyte/platelet development. (A,B) CBC analysis of peripheral blood showed that VavCre+Mef2C^fl/fl mice exhibit marked thrombocytopenia and increased platelet size. Circles indicate analysis of individual mice. PLT indicates platelet count; MPV, mean platelet volume. ***P < .001. (C) Electron microscopy imaging of platelets reveals severe ultrastructural defects of Mef2C-deficient platelets, including enlarged size and loss of elongated form. In addition, Mef2C-deficient platelets demonstrated a loss of α-granules (→) and an increase in depleted granules (dashed arrows). (Images were captured on a Gatan UltraScan 2k*2k camera, developed and scanned at 1200 dpi. Magnification was ×10 000.) Scale bar represents 1 μm. (D) FACS analysis of the HSC/progenitor compartment shows that Mef2C-deficient mice maintain normal phenotypic frequency of HSCs (Lin⁻Sca-1^hickit^hi), and CMP and granulocyte macrophage (GMP) progenitors. A slight increase in the frequency of myelo-erythroid progenitors (MEP) was observed. (E) FACS analysis documents a slight increase of committed unilineage megakaryocytic progenitors (CFU-Mk) in Mef2C-deficient mice. (F) Analysis of the megakaryocyte compartment revealed comparable frequency of immature megakaryocytes (FSC^hiCD41^loCD31⁺) and a slight increase in the frequency of mature megakaryocytes (FSC^hiCD41⁺CD31⁺) in Mef2C-deficient mice. Assessment of ploidy using DRAQ5 showed that Mef2C-deficient immature and mature megakaryocytes are capable of endomitosis (> 4N). FSC^hiCD41⁻CD31⁻ cells are shown as 2N controls. (G) AchE staining of bone marrow cytospins documents the presence of AchE⁺ megakaryocytes in the bone marrow of Mef2C-deficient mice. (Sections were analyzed on a Zeiss Axiovert 40 CFL microscope. Images were captured using a Canon PC1089. Magnification, ×20.) However, after in vitro culture, VavCre⁺Mef2C^fl/fl bone marrow (BM) progenitors displayed a significantly impaired ability to generate AchE⁺ megakaryocytes compared with controls. Bars represent mean plus or minus SD; N = 3. (H) AchE staining of in vitro bone marrow cultures with (Mef2C) or without (mock) transduction with Mef2C retroviral vector shows that Mef2C does not rescue megakaryopoiesis in VavCre⁺Scl^fl/fl bone marrow cultures (i), whereas it does rescue megakaryopoiesis in VavCre⁺Mef2C^fl/fl bone marrow (ii). Bars indicate mean plus or minus SD, n = 4 to 6. **P < .01. ns indicates not significant.