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. 2010 Sep 16;116(11):1942-50.
doi: 10.1182/blood-2010-01-261743. Epub 2010 Jun 4.

Serum response factor is an essential transcription factor in megakaryocytic maturation

Stephanie Halene  1 , Yuan Gao, Katherine Hahn, Stephanie Massaro, Joseph E Italiano Jr, Vincent Schulz, Sharon Lin, Gary M Kupfer, Diane S Krause
Affiliations

Serum response factor is an essential transcription factor in megakaryocytic maturation

Stephanie Halene et al. Blood. .

Abstract

Serum response factor (Srf) is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to Srf(F/F) mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/Srf(F/F) knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41(+) megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.

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Figures

Figure 1
Figure 1
Assessment of megakaryocyte lineage-specific Srf deletion. Bone marrow (BM) from Pf4/Srf+/+ (n = 3) and Pf4/SrfF/F (n = 4) mice was cultured for 4 days in TPO and subjected to BSA gradient separation. Quantitative RT-PCR was performed on the isolated fractions. Mean and SD are shown. P < .01.
Figure 2
Figure 2
Platelet counts, BM progenitor, and Mk ploidy analysis. Blood from wild-type (WT; n = 15), Pf4/SrfF/+ HET (n = 12), and Pf4/SrfF/F knockout (KO; n = 17) mice was analyzed using a Hemavet analyzer. (A) Platelet counts in Pf4/SrfF/F mice were significantly lower than in control mice (P < .001). (B-C) BM from Pf4/Srf WT (n = 8) and KO (n = 11) mice was analyzed by flow cytometry for CD41+ megakaryocytes (B, *P < .001) and progenitor populations (C, **P < .05). (D) BM from WT (n = 3) and KO (n = 4) mice was assayed for CFU-Mk. (E) Plasma TPO levels (in picograms per milliliter) were assayed from Pf4/Srf WT (n = 15) and KO (n = 10) mice. (F) Megakaryocytes from WT (n = 4) and KO (n = 5) mice were analyzed by flow cytometry for nuclear DNA content. *P < .05, **P < .01.
Figure 3
Figure 3
Megakaryocyte morphology and localization. Representative BM (A) and spleen (B) sections of Pf4/Srf WT and KO mice were stained with hematoxylin and eosin (top) and anti-VWF (bottom). Representative images of at least 3 mice are shown.
Figure 4
Figure 4
Megakaryocyte adherence and proplatelet formation. (A-D) Representative images are shown for sorted CD41+c-kit+ megakaryocyte progenitors from Pf4/Srf WT and Pf4/Srf KO littermates that were cultured in TPO containing medium either on fibronectin (left) or in semisolid medium (methylcellulose, right). (E) Continuous videos were taken and analyzed for cell adherence when cultured on fibronectin (left) and for proplatelet formation by cells on fibronectin (middle) and in semisolid medium (right). The percentage of total cells analyzed over 88 hours is shown. **P < .05.
Figure 5
Figure 5
Megakaryocyte, proplatelet, and platelet cytoskeletal structure. Megakaryocytes from Pf4/Srf WT and KO mice were cultured in TPO containing medium for 4 days and subsequently plated on fibronectin-coated coverslips and stained for tubulin (green) and F-actin (phalloidin, red). 4,6′-diamidino-2-phenylindole was used to stain DNA. (A) Representative images of WT (left) and KO (right) megakaryocytes. (B) Representative images of WT (top) and KO (bottom) megakaryocytes forming proplatelets in culture. (C) Platelets from 3 Pf4/Srf WT and 3 Pf4/Srf KO mice were fixed, spun onto coverslips, and stained for tubulin and F-actin. (D) WT (n = 29) and KO (n = 34) platelet size were measured in micrometers. *P < .01.
Figure 6
Figure 6
Transmission electron microscopy of megakaryocytes and platelets. Representative electron microscopic views of platelets and cultured megakaryocytes of Pf4/Srf WT and KO mice. Pf4/Srf KO mice contain a population of giant platelets, and Pf4/Srf KO megakaryocytes have structural alterations. (A) Representative image of a normal population of platelets from a WT mouse. (B-D) Electron micrographs showing giant platelets isolated from the blood of Pf4/Srf KO mice. Scale bar represents 500 nm (A-D). (E) Representative image of a normal megakaryocyte with a well-structured, uniform demarcation membrane system from WT mouse. (F-H) Electron micrographs of megakaryocytes with altered structures from Pf4/Srf KO mice. A small area of demarcation membrane system concentration, resembling a maze of small tubules, is observed in a population of Srf-deficient megakaryocytes (F-G). (H) Representative Pf4/Srf KO megakaryocyte micrographs in which the cellular contents are not evenly distributed throughout the cytoplasm, including areas with missing granules and organelles. Scale bar represents 2 μm (E-H).
Figure 7
Figure 7
Bleeding time and platelet function analysis. (A) Bleeding times were performed on Pf4/Srf WT (n = 8) and KO (n = 8) mice at the time of genotyping. *P < .01. (B-C) Platelet function analysis was performed by activation of platelets with ADP and analysis of FSC/SSC distribution (B) and analysis of GPIIb/IIIa distribution changes by staining with JON/A (C). (D) GPIIb density was confirmed by staining with anti-CD41. Data are representative of at least 3 independent experiments.
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Comment in

  • Megakaryocytes muscle in.
    Tong W. Tong W. Blood. 2010 Sep 16;116(11):1828-9. doi: 10.1182/blood-2010-06-291369. Blood. 2010. PMID: 20847210 No abstract available.

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