MKL1 and MKL2 play redundant and crucial roles in megakaryocyte maturation and platelet formation

Abstract

Serum response factor and its transcriptional cofactor MKL1 are critical for megakaryocyte maturation and platelet formation. We show that MKL2, a homologue of MKL1, is expressed in megakaryocytes and plays a role in megakaryocyte maturation. Using a megakaryocyte-specific Mkl2 knockout (KO) mouse on the conventional Mkl1 KO background to produce double KO (DKO) megakaryocytes and platelets, a critical role for MKL2 is revealed. The decrease in megakaryocyte ploidy and platelet counts of DKO mice is more severe than in Mkl1 KO mice. Platelet dysfunction in DKO mice is revealed by prolonged bleeding times and ineffective platelet activation in vitro in response to adenosine 5'-diphosphate. Electron microscopy and immunofluorescence of DKO megakaryocytes and platelets indicate abnormal cytoskeletal and membrane organization with decreased granule complexity. Surprisingly, the DKO mice have a more extreme thrombocytopenia than mice lacking serum response factor (SRF) expression in the megakaryocyte compartment. Comparison of gene expression reveals approximately 4400 genes whose expression is differentially affected in DKO compared with megakaryocytes deficient in SRF, strongly suggesting that MKL1 and MKL2 have both SRF-dependent and SRF-independent activity in megakaryocytopoiesis.

Figure 1

MKL2 gene expression and validation of conditional Mkl2 KO mice. (A) MKL1 and MKL2 mRNA levels were assessed in PreMegE cells from 3 WT mice differentiated in vitro using megakaryocyte differentiation medium. Shown is the fold increase in mRNA over freshly sorted PreMegE of megakaryocytes from 5-day cultured PreMegE cells after normalization to the 18S internal control. All 3 mice show an increase in both MKL1 and MKL2 during megakaryocyte differentiation. (B) Mkl2 expression was assessed in megakaryocytes differentiated in vitro from PreMegE of WT (n = 3) and Mkl1 KO (n = 3) mice. Shown is the fold increase in mRNA over HSC. Error bars represent SEM. (C) PCR of genomic DNA isolated from HSC, PreMegE, and MkP after 3 days of mTPO culture showed specific deletion of the Mkl2 locus in megakaryocytes of Pf4-Cre expressing Mkl2^F/F mice. Mkl2^F/F mice without Pf4-Cre were negative controls.

Figure 2

DKO mice have macrothrombocytopenia and dysfunctional platelets. Peripheral blood was taken from mice with the indicated genotypes and (A) platelet counts and (B) platelet volume analyzed. (C) Representative peripheral blood smears stained with Wright Giemsa are consistent with low platelet count and high MPV in DKO mice. Images were taken using an oil-immersion 100× lens. Black arrows indicate platelets. (D) Bleeding times from mice with different genotypes (WT, n = 31; Mkl1 KO, n = 6; Mkl2 cKO, n = 10; DKO, n = 13). (E) Flow cytometry of peripheral blood platelets showing FSC vs SSC in the absence (top) and presence (bottom) of ADP. Note change in shape of platelet gate (circled in red) in response to ADP stimulation. Red blood cells (RBCs) are indicated. (F) Representative data showing total CD41/61 (x-axis) versus the activated JON/A conformation (y-axis) of CD41/CD61 in resting (blue) and ADP treated (red) platelets of 4- to 6-week-old mice. (n.s. indiates not significant; ****P < .0001; ***P < .001; **P < .01). All error bars represent SEM. Data from 4 independent experiments are summarized in Table 1.

Figure 3

DKO platelets lack normal cytoskeleton organization and granule complexity. Platelet-rich plasma was isolated from mouse blood. (A) Samples were spun onto poly-l-lysine–coated slides and fixed immediately or permitted to spread for 20 minutes on glass before fixation. Samples were permeabilized and probed for filamentous actin (red, phalloidin) and β1 tubulin (green). Quantification of phalloidin intensity by immunofluorescence showed decreased polymerized actin in Mkl1 KO and DKO platelets (bottom). Error bars represent SEM. (B) Thin-section electron micrographs highlight the heterogeneity in granule segregation and platelet morphology. DKO platelets lack α granules and their dense granules are not as opaque. Red squares in center panels indicate the magnified sections in the right panels. Magnified right panel images were modified for easier visualization of microtubule cross-sections by increasing contrast. Thick arrows indicate marginal band microtubules, which are increased in DKO mice (***P < .001).

Figure 4

DKO mice have an accumulation of immature megakaryocytes in the BM. Paraffin sections from femurs of 6-week-old mice were stained with (A) anti-VWF antibody or (B) H&E. (C) Representative ploidy histograms for CD41⁺ bone marrow cells are shown along with the mean ploidy (MP) ± SEM of 4 mice per genotype. (D) Consistent with the decreased mean ploidy, the percentages of megakaryocytes with each ploidy level for n = 4 mice per genotype show that Mkl1 KO and DKO megakaryocytes have a significant increase in 2N megakaryocytes. (E) Flow cytometry confirms the increase in total CD41⁺ cells in the bone marrow using 4 mice per genotype. (F) Analysis of BM progenitors revealed an increase in the PreMegE and MkP populations in DKO BM (n.s. indicates not significant; **P < .01; *P < .05; all error bars represent SEM).

Figure 5

Abnormal cytoskeleton in DKO megakaryocytes. (A) Representative thin-section electron micrographs of fetal liver–derived megakaryocytes from (i,ii) WT and (iii,iv) DKO embryos. (B) Phalloidin staining of fetal liver–derived megakaryocytes (top) and quantification of F-actin (bottom) show decreased polymerized actin in DKO (***P < .001; all error bars represent SEM).

Figure 6

DKO and Srf Pf4-cKO megakaryocytes have distinct gene expression profiles. (A) Heat maps displaying the differential gene expression patterns of megakaryocytes from the indicated genotypes. Red color represents elevated expression while green represents decreased expression compared with the row mean. Genes displayed were selected based on fold changes of 2 or more and FDR adjusted P value < .05 between WT and DKO. (C) Venn diagrams showing genes with fold changes of 2 or more and FDR adjusted P value < .05 for the indicated comparisons. Representative qPCR expression of EGFL7, miR-126, and SPRED1 in megakaryocytes. Values are displayed as log₂ fold change over WT.