MKL1 and MKL2 Play Redundant and Crucial Roles in Megakaryocyte Maturation and Platelet Formation

From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

PLATELETS AND THROMBOPOIESIS

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

Elenoe C. Smith,1 Jonathan N. Thon,2 Matthew T. Devine,2 Sharon Lin,3 Vincent P. Schulz,4 Yanwen Guo,5 Stephanie A. Massaro,4 Stephanie Halene,6 Patrick Gallagher,4,5 Joseph E. Italiano Jr,2,7 and Diane S. Krause1,3

1Department of Cell Biology, Yale University School of Medicine, New Haven, CT; 2Hematology Division, Department of Medicine, Brigham and Women’s Hospital, Boston, MA; Departments of 3Laboratory Medicine, 4Pediatrics, 5Genetics, and 6Hematology, Yale University School of Medicine, New Haven, CT; and 7Vascular Biology Program, Department of Surgery, Children’s Hospital, Boston, MA

Serum response factor and its transcrip- ploidy and platelet counts of DKO mice is than mice lacking serum response factor tional cofactor MKL1 are critical for mega- more severe than in Mkl1 KO mice. Plate- (SRF) expression in the megakaryocyte karyocyte maturation and platelet forma- let dysfunction in DKO mice is revealed compartment. Comparison of gene ex- tion. We show that MKL2, a homologue of by prolonged bleeding times and ineffec- pression reveals approximately 4400 genes MKL1, is expressed in megakaryocytes tive platelet activation in vitro in response whose expression is differentially affected -and plays a role in megakaryocyte matura- to adenosine 5؅-diphosphate. Electron mi- in DKO compared with megakaryocytes de tion. Using a megakaryocyte-specific Mkl2 croscopy and immunofluorescence of ficient in SRF,strongly suggesting that MKL1 knockout (KO) mouse on the conven- DKO megakaryocytes and platelets indi- and MKL2 have both SRF-dependent and tional Mkl1 KO background to produce cate abnormal cytoskeletal and mem- SRF-independent activity in megakaryocyto- double KO (DKO) megakaryocytes and brane organization with decreased gran- poiesis. (Blood. 2012;120(11):2317-2329) platelets, a critical role for MKL2 is re- ule complexity. Surprisingly, the DKO mice vealed. The decrease in megakaryocyte have a more extreme thrombocytopenia Introduction

Biphenotypic megakaryocyte-erythroid precursors undergo embryos dying because of myocardial cell necrosis. However, differentiation and endomitosis to become mature polyploid mega- Mkl1 KO mice that complete gestation have normal lifespans. karyocytes that release platelets into the circulation. Despite Functions for MKL1 have been characterized in embryonic stem advances in our understanding of hematopoiesis, much remains cells, fibroblasts, smooth muscle cells, and neurons.10-12 unknown regarding the regulation of megakaryocytopoiesis. Our Recently, we and others characterized the hematopoietic pheno- laboratory is focused on understanding the mechanisms of normal types of Mkl1 KO and megakaryocyte-specific Srf conditional KO megakaryocyte differentiation to better understand acute megakaryo- (Srf Pf4-cKO) mice.13-15 In each case, mice have thrombocytopenia blastic leukemia (AMKL). The reciprocal t(1;22) translocation that with increased numbers of immature megakaryocytes in the bone is consistently associated with AMKL results in fusion of the marrow (BM) suggesting both a failure of normal megakaryocyte RBM15 (RNA-binding motif 15) and MKL1 (megakaryoblastic maturation as well as abnormal formation of the megakaryocyte leukemia 1) genetic loci.1,2 RBM15, an RNA-binding protein and platelet cytoskeleton. Comparison of the Mkl1 KO and Srf whose function is not yet well defined, is differentially expressed in Pf4-cKO mice as well as in vitro cell-culture studies suggest that hematopoiesis with the highest mRNA levels in progenitors and the effect of MKL1 in megakaryocyte differentiation is mediated lowest in differentiated blood cells.3 Here, we focus on the role of by association with SRF.13 However, the hematopoietic phenotype the MKL1 family of proteins in megakaryocytopoiesis. is far more severe in Srf Pf4-cKO than Mkl1 KO mice, which MKL1 (MRTF-A, MAL, BSAC) is a transcriptional cofactor indicates that other factors may act in conjunction with, or in the belonging to the myocardin-related transcription factor (MRTFs) absence of, MKL1 to promote SRF-mediated megakaryocyte family that also includes myocardin and MKL2 (MRTF-B, maturation. Possible contributing proteins include other members MAL16). These proteins function through association with of the MRTF family. serum response factor (SRF), a ubiquitously expressed transcrip- Here, we assessed the contribution of MKL2 to megakaryocyto- tion factor.4 SRF is also activated by association with ternary poiesis, and found that while it is not required for effective complex factors (Elk1, SAP1, SAP2).5 Transcriptional cofactor megakaryocyte maturation and platelet formation, MKL2 compen- binding determines the spatiotemporal activity of SRF. Srf sates for and mitigates the effects of MKL1 deficiency. Mice knockout (KO) mice die early in gestation because of abnormal lacking both MKL1 and MKL2 in the megakaryocyte lineage mesoderm development.6 Myocardin and Mkl2 KO mice are also have macrothrombocytopenia as well as platelet cytoskeletal embryonic lethal because of abnormal cardiac development.7-9 abnormalities and severely impaired platelet activation. In addition Mkl1 KO mice show partial embryonic lethality, with some to the peripheral blood defects, there is abnormal megakaryocyte

Submitted March 30, 2012; accepted July 6, 2012. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, July 17, 2012; DOI 10.1182/blood-2012-04-420828. payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734. The online version of this article contains a data supplement. © 2012 by The American Society of Hematology

BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11 2317 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2318 SMITH et al BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11 ultrastructure. Surprisingly, the phenotype of double KO (DKO) previously described.20 Samples were examined with an Axiovert 200 mice is more extreme than that of the Srf Pf4-cKO mice, strongly microscope (Carl Zeiss Inc) equipped with a 63ϫ NA 1.4 oil-immersion suggesting that MRTFs function in ways that are independent of objective. Images were obtained using a charge-coupled device camera their SRF transcriptional coactivation activities. (Hamamatsu Photonics) and phalloidin quantiﬁed using NIH ImageJ software.

Methods DNA and RNA analysis DNA and RNA were extracted using the QIAGEN DNeasy Blood and Mouse strains Tissue and Ambion RNAqueous Micro kits, respectively. cDNA was made All procedures were performed in compliance with relevant laws and using Superscript III (Life Technologies) with random primers (Life institutional guidelines and were approved by the Yale University Institu- Technologies). Quantification was performed using a CFX96 C1000 tional Animal Care and Use Committee. Mkl2F/F mice16 were crossed with thermal cycler (Bio-Rad) using TaqMan gene expression assays (Applied platelet factor 4 (Pf4) Cre17-expressing mice. These mice were then bred Biosystems–Life Technologies): murine epidermal growth factor-like do- onto the Mkl1 KO background.7 Mkl1 KO and Pf4-Cre mice crossed with main 7 (EGFL7) and murine Spred1, and eukaryotic 18S as an internal SrfF/F were as previously described.13,14 All mice were on a C57Bl/6J control. U6 RNA was used as an internal control for miR-126 as assayed by background. QIAGEN miScript SYBR Green primer assay. MkP were sorted from the BM of 5- to 10-week-old mice and RNA harvested after cells were differentiated for 3 days in megakaryocyte differentiation media. Samples Flow cytometry, cell sorting, and in vitro culture were collected from at least 8 different mice for each genotype and pooled To determine mouse BM progenitor populations, freshly isolated BM was for RNA sequencing. Library preparation and sequencing were performed incubated with anti-CD16/CD32 antibodies (Fc block; BD Biosciences), by the Yale Stem Cell Genomics Core Facility using the Illumina TruSeq stained with PE-biotin lineage detection cocktail (Miltenyi Biotec), RNA Sample Preparation kit. Samples were sequenced on an Illumina allophycocyanin-H7 CD117/c-kit (BD Biosciences), Alexa 647 Sca-1, HiSeq 2000 using 50-cycle single-end sequencing. FASTQ format sequenc- PE-Cy5 CD150, PE-Cy7 CD105 (BioLegend), and FITC CD41 (BD ing reads were aligned to the mm9 genome using Tophat Version 1.3.1 Biosciences), and analyzed (LSRII; BD Biosciences) or sorted (FACS Aria, software.21 The cufflinks, cuffmerge, and cuffdiff Version 1.3.0 programs BD Biosciences; or MoFlo, Beckman Coulter) as previously described.18 were used to identify differences in Ensembl transcripts.22 The analysis For sorting experiments, BM was first enriched for stem/progenitor cells used upper-quartile normalization, multiread, and GC fragment bias using immunomagnetic separation (BD Biosciences). LSK (lineage [lin] corrections, and masking of reads in rRNA and tRNA genes. Sample negative, CD117 positive, Sca-1 positive), PreMegE (lin negative, CD117 comparisons are displayed using the R heatmap.2, lumi, and VennDiagram positive, CD41 negative, CD150 positive, CD105 negative), and MkP (lin packages. Data are publicly available through Gene Expression Omnibus negative, CD117 positive, CD41 positive) were assayed. For DNA content (file numbers pending). Statistically significant differences between geno- analysis, unfractionated whole BM was stained with FITC CD41, then types were defined by genes having more than 10 fragments per kilobase of treated with 70% ethanol overnight followed by digestion with 20 ␮g/mL transcript per million (FPKM) reads in at least 1 of the 2 samples compared RNase (Sigma-Aldrich) on ice for 4 hours. Cells were resuspended with and a q value of less than 0.05. 10 ␮g/mL propidium iodide (Sigma-Aldrich) before analysis on a FACSCali- bur (BD Biosciences). Platelet activation was assessed using FITC CD41/61 Bone marrow histology and PE JON/A (Emfret Analytics). Flow cytometric data were analyzed using FlowJo software (TreeStar). PreMegE and MkP were differentiated Femurs were fixed overnight in 4% paraformaldehyde, decalcified in for 5 or 3 days, respectively, in megakaryocyte differentiation medium Decalcifier I solution (Surgipath) overnight, and transferred to 70% ethanol containing StemSpan Serum Free Expansion Media (StemCell Technolo- for processing by the Research Histology Facility at Yale School of gies) supplemented with 30% BIT 9500, L-glutamine (Life Technologies), Medicine for 5-␮m longitudinal paraffin sections, hematoxylin and eosin penicillin/streptomycin (P/S; Life Technologies), and 50 ng/mL murine (H&E) staining, and immunohistochemistry for von Willebrand factor thrombopoietin (mTPO; ConnStem). Fetal liver megakaryocytes were (anti-VWF; DAKO). obtained from E13.5 embryos and cultured for 4 days in low-glucose DMEM (Life Technologies), 10% fetal bovine serum (Gemini), P/S, and 50 ng/mL mTPO. Bleeding time measurements Three-week-old mice were anesthetized using isoflurane (Butler Animal Platelet preparations Health Support). Using a sharp razor blade, 0.5 cm of the tail was removed and the tail held in warm PBS. Bleeding time was measured as the time until Peripheral blood was collected from the retro-orbital sinus into tubes bleeding stopped. containing acid citrate dextrose (ACD) anticoagulant. Platelet-rich plasma was prepared as previously described.19 For flow cytometry, 50 ␮Lof whole blood was added to 200 ␮L of 20 U/mL heparin in Tris-buffered Statistical analysis saline (20mM Tris-HCl, 137mM NaCl). After further dilution with 1 mL of Statistical significance was assessed using the 2-tailed unpaired t test with 2mM CaCl in modified Tyrode-HEPES buffer (5mM HEPES, 140mM 2 Prism software (GraphPad). NaCl, 2.7mM KCl, 5.5mM dextrose, 0.42mM Na2HPO4, 12mM NaHCO3), platelets were stimulated with 1mM adenosine 5Ј-diphosphate (ADP; Sigma-Aldrich). Results

Immunofluorescence MKL2 is expressed in murine megakaryocytes Platelets isolated from peripheral blood or fetal liver–derived megakaryocytes were spun onto poly-L-lysine–coated coverslips and either The hematopoietic phenotype of megakaryocyte-specific (Pf4-Cre) immediately fixed with 2% paraformaldehyde or allowed to spread for Srf conditional KO (Srf Pf4-cKO) is more severe than that of Mkl1 14 20 minutes and then fixed (platelets only). Permeabilization, blocking, KO mice, suggesting compensation or redundancy in Mkl1 KO and staining with using a ␤1 tubulin antibody (Genemed Synthesis Inc) mice. Because there are multiple transcriptional coactivators of and Alexa Fluor 568 Phalloidin (Life Technologies) were conducted as SRF, multiple other proteins may mitigate the effects of MKL1 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11 MKL2 IN MEGAKARYOCYTOPOIESIS 2319

Conﬁrmation of Mkl2 conditional knockout mice in the megakaryocyte lineage

To assess the function of MKL2 in megakaryocytes, mice with megakaryocyte-specific KO of MKL2 (Mkl2 Pf4-cKO) were made by crossing Mkl2F/F mice with platelet factor 4 (Pf4)–Cre mice. In the Mkl2F/F mouse created by the Olson laboratory,16 the floxed region of the Mkl2 gene includes exon 8, which encodes the SRF-binding domain. Mkl2 Pf4-cKO mice are viable. Megakaryocyte-specific deletion was confirmed by genomic PCR of 3 cell populations: MkP cultured for 3 days in megakaryocyte differentiation media, freshly isolated hematopoietic stem/ progenitor cells (LSK), and PreMegE (Figure 1C). As expected, deletion of Mkl2 was not detected in LSK or PreMegE populations, neither of which has yet committed to the megakaryocytic lineage. In contrast, the genome of the differentiated MkP from Pf4-Cre mice contained copies of the excised allele and not the floxed allele (Figure 1C).

Conditional DKO mice have thrombocytopenia with defective platelet activation

The Mkl2 Pf4-cKO mice were mated onto the Mkl1 KO background (referred to as “DKO mice”) to create DKO megakaryocytes. The DKO mice have macrothrombocytopenia; platelet counts of DKO mice are significantly (P Ͻ .0001) decreased (187 000 Ϯ 56 000 platelets/␮L) compared with WT (730 000 Ϯ 148 000 platelets/␮L), Mkl1 KO (460 000 Ϯ 94 000 platelets/␮L), and Mkl2 Pf4-cKO (715 000 Ϯ 120 000 platelets/ ␮L; Figure 2A). In the absence of MKL2, 1 copy of MKL1 is sufficient to maintain normal platelet counts (643 000 Ϯ 157 000 platelets/␮L). In contrast, mice with 1 copy of MKL2 in the absence of MKL1 have platelet counts (325 000 Ϯ 54 000 platelets/ ␮L) significantly higher than DKO mice, but lower than WT mice. Srf Pf4-cKO platelet numbers (416 000 Ϯ 74 000 platelets/␮L) were decreased compared with WT, consistent with previous reports,14 however, they were significantly higher than DKO mice. 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 DKO mice also had increased mean platelet volume (MPV; differentiated in vitro using megakaryocyte differentiation medium. Shown is the fold Figure 2B) as assessed by peripheral blood smears (Figure 2C). increase in mRNA over freshly sorted PreMegE of megakaryocytes from 5-day Further characterization of the DKO animals indicated defec- cultured PreMegE cells after normalization to the 18S internal control. All 3 mice show Ϯ an increase in both MKL1 and MKL2 during megakaryocyte differentiation. (B) Mkl2 tive hemostasis. Bleeding times of the DKO mice (171 86 sec- expression was assessed in megakaryocytes differentiated in vitro from PreMegE of onds) were significantly increased over wild-type (WT; 35 Ϯ 14 sec- ϭ ϭ WT (n 3) and Mkl1 KO (n 3) mice. Shown is the fold increase in mRNA over onds), Mkl1 KO (73 Ϯ 52 seconds), and Mkl2 Pf4-cKO 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 (34 Ϯ 13 seconds) mice (Figure 2D). It is not clear why 3 of Mkl2 locus in megakaryocytes of Pf4-Cre expressing Mkl2F/F mice. Mkl2F/F mice 13 DKO mice had bleeding times within the normal range. The without Pf4-Cre were negative controls. prolonged bleeding times of DKO mice could be because of decreased platelet counts and/or platelet dysfunction. In vitro platelet function was assayed by appearance of activated CD41/ CD61 and morphologic changes in response to ADP. In the resting deficiency. Possible genes that could compensate for the loss of state, WT, Mkl1 KO, Mkl2 Pf4-cKO, and Srf Pf4-cKO platelets MKL1 are Myocardin and Mkl2. Myocardin’s expression is displayed normal forward scatter (FSC) and side scatter (SSC). In restricted to the heart. We investigated whether MKL2, known to be expressed in multiple cell types, is expressed in megakaryo- contrast, DKO platelet morphology was less uniform and the cytes. RNA was taken from PreMegE cells sorted by flow platelet and red cell populations were not well separated (Fig- cytometry immediately postsort (day 0) and after culturing 5 days ure 2E, Table 1), consistent with increased MPV. After ADP in megakaryocyte differentiation media. MKL2 expression in- treatment, there was an increase in activated CD41/61 heterodimer creased during megakaryocyte differentiation, although not as on the surface of WT, Mkl1 KO, and Mkl2 Pf4-cKO platelets using dramatically as MKL1 (Figure 1A). We hypothesized that Mkl2 a PE-conjugated JON/A antibody (Figure 2F, Table 1). In contrast, mRNA would be up-regulated to compensate for the lack of MKL1 DKO platelets did not respond to ADP; there was no conforma- in the Mkl1 KO mice. However, there was no increase in Mkl2 tional change of the CD41/61 heterodimer to allow for JON/A expression in megakaryocytes derived from PreMegE cells in Mkl1 antibody binding. In addition, DKO platelets did not display any KO mice compared with WT controls (Figure 1B). shape change within the FSC/SSC gate after ADP treatment. From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2320 SMITH et al BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11

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 100ϫ lens. Black arrows indicate platelets. (D) Bleeding times from mice with different genotypes (WT, n ϭ 31; Mkl1 KO, n ϭ 6; Mkl2 cKO, n ϭ 10; DKO, n ϭ 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 Ͻ .0001; ***P Ͻ .001; **P Ͻ .01). All error bars represent SEM. Data from 4 independent experiments are summarized in Table 1.

DKO platelets have cytoskeleton abnormalities and atypical eton organization in DKO platelets as assayed by phalloidin and morphology ␤1-tubulin, a megakaryocyte lineage-specific isoform of tubulin Flow cytometry suggested altered platelet structure and shape. (Figure 3A). Resting WT and Mkl2 Pf4-cKO platelets showed Because many cytoskeleton genes are SRF targets, organization of significant filamentous actin staining (shown in red) and distinct polymerized actin and microtubules of the cytoskeleton were microtubule rings (shown in green) characteristic of platelets assessed. Immunofluorescence images of platelets, both resting and (Figure 3Ai,iii). Mkl1 KO platelets had an organized microtubule spread, showed differences in the actin and microtubule cytoskel- ring but lacked phalloidin staining (Figure 3Aii). After contact with From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

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Table 1. Platelet activation ﬂow cytometry parameters Resting ADP activated WT MKL1 KO MKL2 cKO DKO WT MKL1 KO MKL2 cKO DKO

Mean FSC 28 Ϯ .32 25 Ϯ .84 29 Ϯ .42 47 Ϯ 3.9* 30 Ϯ .53 29 Ϯ .46 33 Ϯ 1.2 52 Ϯ 4.9* JON/A, MFI 17 Ϯ .4 16 Ϯ .26 17 Ϯ .25 20 Ϯ 1.0* 106 Ϯ 16 104 Ϯ 11 108 Ϯ 48 30 Ϯ 1.4*

Mean FSC and MFI of JON/A staining was averaged and the SD displayed (n ϭ 4). ADP indicates adenosine 5Ј-diphosphate; WT, wild type; KO, knockout; cKO, conditional knockout; DKO, double knockout; FSC, forward scatter; and MFI, mean fluorescence intensity. *P Ͻ .001. glass, WT and Mkl2 Pf4-cKO platelets retract their tubulin coils, retained partial microtubule coil morphology and the actin cytoskel- and polymerize actin to extend filopodia and lamellopodia, ulti- eton remained disorganized (Figure 3Avi). DKO platelets did not mately causing them to spread (Figure 3Av,vii). Although spread change shape after adhesion to coverslips and had no filamentous Mkl1 KO platelets had some microtubule reorganization, most actin staining (Figure 3Aviii). In addition to the abnormal platelet

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 ␤1 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 ␣ 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 Ͻ .001). From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2322 SMITH et al BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11 activation, DKO platelets were often amorphously shaped, unlike (P Ͻ .05) increase over WT cells only in the MkP and total CD41ϩ the characteristic discoid shape of WT platelets. Quantification of populations. DKO megakaryocytes have a statistically significant phalloidin by immunofluorescence confirmed the decrease in increase in 4N megakaryocytes at the expense of Ն 8N megakaryo- polymerized actin (Figure 3A bottom panels). Of the DKO plate- cytes compared with Mkl1 KO mice; this suggests that DKO lets with detectable ␤1 tubulin, the marginal tubulin coil was mice have more impaired megakaryocyte maturation than Mkl1 thicker than their WT and single KO counterparts and revealed KO mice. distinct heterogeneity in size and shape (Figure 3Aiv). The bright ␤1-tubulin immunofluorescence signal in the DKO platelets also Megakaryocytes from DKO mice have abnormal cytoplasmic suggested an increase in ␤1-tubulin protein. ultrastructure As suggested by flow cytometry (Figure 2E), DKO platelets Ultrastructural analysis revealed morphologic abnormalities in have less uniform granularity and size than WT, Mkl1 KO, and DKO megakaryocytes (Figure 5A). Compared with WT, DKO Mkl2 Pf4-cKO platelets. To analyze this defect in more detail, megakaryocytes have cytoplasmic regions that are devoid of platelet ultrastructure was assessed by electron microscopy. The demarcation membrane and increased numbers of vacuoles, which images shown highlight the defective cytoskeleton and abnormal normally appear during later stages of maturation (Figure 5Aiii,iv). granular contents of the DKO platelets (Figure 3B). A unique In addition, the areas of microvesiculation that occur around the feature of platelets is the microtubule ring that lies just below the plasma membrane of WT megakaryocytes were present throughout platelet surface. This ring is a single microtubule, looped 8-12 times, the cytoplasm of DKO megakaryocytes in the process of proplate- allowing a 100-micron tubule to exist within a 2- to 4-micron let formation and may account for the heterogeneity in platelet platelet, giving platelets their characteristic discoid shape. Electron shape, size, and granule content observed in these mice micrograph analysis of WT, Mkl1 KO, and Mkl2 Pf4-cKO platelets (Figure 5Aiii,iv). Analysis of Srf Pf4-cKO mice suggests that revealed normal numbers of microtubule rings in cross-section8-12 defects in the actin cytoskeleton are responsible for the abnormal at their tips while DKO platelets were seen to have more (up to 17) megakaryocyte and platelet morphology.14 Measurement of the microtubules when cross-sectioned (Figure 3B thick black arrows). filamentous actin was determined by quantitative immunofluores- These data correlate with the increased intensity of the ␤1-tubulin cence microscopy of phalloidin (Figure 5B), which showed a immunostaining (Figure 3Aiv,viii). In general, the DKO platelets significant decrease in actin organization in the DKO megakaryo- were amorphously shaped with poorly defined borders, unlike the cytes, consistent with the hypothesis that the actin cytoskeleton is uniformly discoid shape of the WT platelets. Most notable, disordered. however, was the loss of heterogeneity of the granular contents of the DKO platelets. Some of the DKO platelets had granular RNA sequencing reveals SRF-independent functions for distribution very similar to WT, while others lacked both ␣ and MRTFs in megakaryocyte differentiation dense granules. Investigation of the role of MKL2 in megakaryocytopoiesis DKO BM has an accumulation of immature megakaryocytes suggests that MKL2 functions in the absence of MKL1 to promote Platelet defects in peripheral blood suggest abnormal BM mega- megakaryocyte maturation, and that much of this effect is mediated karyocytopoiesis. Immunohistochemistry for VWF on BM sections via activation of SRF target genes. However, while the data (Figure 4A) revealed an increase in megakaryocyte number in indicate that many of the defects in DKO mice phenocopy those of DKO, and, to a lesser extent, Mkl1 KO mice (already reported), SRF cKO mice, the platelet counts of DKO mice are more compared with WT and Mkl2 Pf4-cKO BM. The VWF-positive dramatically reduced. This may be because of SRF-independent megakaryocytes in the DKO were often small, with low ploidy and functions of the MRTFs. To assess these novel functions, we irregular cell morphology, consistent with the staining patterns sequenced the transcriptomes of WT, Mkl1 KO, Mkl2 Pf4-cKO, found in Srf Pf4-cKO mice. H&E staining did not reveal any DKO, and Srf Pf4-cKO MkPs differentiated in vitro for 3 days with differences in BM cellularity (Figure 4B). TPO. We confirmed that the population of differentiated megakaryo- Flow cytometric analysis of DKO BM CD41ϩ cells showed a cytes analyzed from each genotype consisted of a relatively pure Ͼ ϩ ϩ statistically significant (P Ͻ .01) shift to lower ploidy megakaryo- ( 93%) CD41 /CD42 cell population without myeloid contami- cytes compared with WT (Figure 4C-D). Ploidy measures the nation (data not shown). DNA content of cells, thus assessing the number of endomitotic Analysis of the deep sequencing data revealed that DKO cycles a cell has undergone. Mkl1 KO and DKO BM had a megakaryocytes had the most altered expression profile compared similar percentage of megakaryocytes that were 2N. However, with the other 4 genotypes. A majority of the pairwise comparisons there were fewer DKO megakaryocytes with high ploidy (Ն 8N) did not have many genes with greater than 2-fold differential gene compared with Mkl1 KO, resulting in a statistically significant expression (supplemental Figure 1, available on the Blood Web increase in 4N cells in DKO compared with Mkl1 KO mice. This site; see the Supplemental Materials link at the top of the online suggests that MKL2 may play a role in promoting the second article). A total of 969 genes showed a Ͼ 2-fold difference between and subsequent endomitotic cycles as opposed to the first 2N to WT and Srf Pf4-cKO, while 2906 genes had a Ͼ 2-fold difference 4N endomitosis. There was a statistically significant decrease in between WT and DKO megakaryocytes. Between Srf Pf4-cKO and mean ploidy of DKO CD41ϩ cells (n ϭ 4) compared with WT DKO megakaryocytes, there were 3298 genes with a Ͼ 2-fold (n ϭ 4; P Ͻ .001) and Mkl1 KO (n ϭ 4; P Ͻ .05) megakaryo- change in expression (Figure 6A-B, supplemental Figure 1). As cytes. Consistent with the lack of a platelet phenotype, Mkl2 expected, the DKO megakaryocytes significantly differed from Pf4-cKO CD41ϩ BM cells did not show any difference from WT Mkl1 KO megakaryocytes and consistent with the more extreme BM. The increased number of DKO BM megakaryocytes was phenotype of the DKO mice, DKO megakaryocyte gene expression confirmed by flow cytometry (Figure 4E); DKO marrow also had was more dissimilar to WT than any other genotype (Figure 6A, greater percentages of PreMegE and MkP versus WT (Figure 4F). supplemental Figure 1). Although there were many genes that As shown before,13,15 Mkl1 KO mice had a statistically significant showed similar changes in gene expression in WT compared with From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11 MKL2 IN MEGAKARYOCYTOPOIESIS 2323

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 CD41ϩ bone marrow cells are shown along with the mean ploidy (MP) Ϯ SEM of 4 mice per genotype. (D) Consistent with the decreased mean ploidy, the percentages of megakaryocytes with each ploidy level for n ϭ 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 CD41ϩ 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 Ͻ .01; *P Ͻ .05; all error bars represent SEM).

DKO and Srf Pf4-cKO, most genes were DKO-specific (Figure 6B). The differentially expressed genes open several interesting The appearance of DKO-specific gene expression differences is avenues of research that give insight into the cellular properties unlikely because of incomplete removal of SRF in Srf Pf4-cKO, that drive the dramatic DKO platelet phenotype. We mined the since there are also many genes that differ between Srf Pf4-cKO RNA sequencing data for genes already defined by others as and WT, but not between DKO and WT (Figure 6B and data not megakaryocyte-specific and genes highly expressed during shown). As expected, genes involved in cytoskeletal organization megakaryocyte differentiation.23,24 Of the 56 genes curated by were significantly decreased in Mkl1 KO, DKO, and Srf Pf4-cKO the Papoutsakis group as being megakaryocyte related by megakaryocytes compared with WT (Table 2). literature review, 24 were deregulated by loss of both MKL1 and From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2324 SMITH et al BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11

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 Ͻ .001; all error bars represent SEM).

MKL2 while only 11 of those same genes were affected by loss of SRF (Table 3). Of 58 transmembrane proteins enriched in Discussion megakaryocytes, 22 had altered gene expression in DKO megakaryocytes and 14 were deregulated in SRF Pf4-cKO Megakaryocytes lacking expression of MKL1 and MKL2 have megakaryocytes, while none of the 58 genes was significantly both defective megakaryocytopoiesis and thrombopoiesis. Mkl1 affected by loss of MKL1 alone (Table 4). Slc35d3, one of the KO mice with megakaryocyte-specific loss of MKL2 have in- genes that is significantly decreased uniquely in DKO cells, is creased megakaryocyte progenitors and immature megakaryocytes essential for platelet dense granule biogenesis.25,26 Of the in the BM. DKO mice have macrothrombocytopenia and increased 35 genes that were up-regulated at least 16-fold compared with bleeding times as a result of ineffective megakaryocytopoiesis and WT in both the Srf cKO and DKO (Table 5), 20 are strongly defects in platelet formation and activation. Many, but not all, of associated with defense response, particularly neutrophil and the differences between the Mkl1 KO and the SRF Pf4-cKO can be mast cell differentiation (for example, C/EBP epsilon). These explained by the presence of MKL2. data suggest that SRF, via activation by MRTFs, may act as a This study is the first to describe a role for MKL2 in master switch to turn off myeloid differentiation programs hematopoiesis. Although MKL2 was known to function in the brain during megakaryocyte commitment. and smooth muscle cells,8,16,31 investigators had dismissed MKL2 Epidermal growth factor-like domain 7 (Egfl7) was the most as a mediator of megakaryocytopoiesis because expression in significantly overexpressed gene in DKO compared with WT, human megakaryocytes is low.32 While our mouse studies also Mkl1 KO, Mkl2 Pf4-cKO, and Srf Pf4-cKO megakaryocytes. Its show that MKL2 is not as highly expressed as MKL1, expression was up-regulated 10-fold in DKO megakaryocytes by megakaryocyte-specific deletion of MKL2 in the absence of MKL1 RNA sequencing analysis and greater than 6-fold by quantitative indicates that MKL2 plays a role, albeit redundant, in megakaryo- PCR analysis (Figure 6C and data not shown). Egfl7 protein is cytopoiesis. SRF target genes that are differentially expressed in expressed and secreted by endothelial cells, and Egfl7 knock- the absence of SRF, MKL1, or MKL2 have been determined by this down in primary human endothelial cells results in decreased study and others.14,15 Future studies will be focused on determining proliferation and migration.27 Within Egfl7’s seventh intron is how these differentially expressed target genes cause the differ- miR-126. Consistent with the Egfl7 data, expression of miR-126 ences in phenotypes between the DKO and Srf Pf4-cKO megakaryo- as determined by quantitative PCR is also significantly increased cytes and platelets. To further determine the mechanisms by which in DKO megakaryocytes (Figure 6C). Gene set enrichment MKL1 and MKL2 act to promote megakaryocytopoiesis, it will be analysis28,29 confirmed the decrease in expression of miR-126 essential to know where SRF, MKL1, and MKL2 are bound on the target genes in DKO megakaryocytes compared with WT mega- chromatin during megakaryocyte differentiation. Furthermore, it karyocytes (supplemental Figure 2). RNA sequencing and qPCR will be important to determine whether the ability of MKL2 to of DKO megakaryocytes showed decreased expression of Spred1, compensate for MKL1 is because of MKL2 occupation of the a published target of miR-12630 (Figure 6C and data not shown). genomic sites left vacant by the MKL1 deficiency in an Mkl1 KO Therefore, the more extreme megakaryocyte and platelet pheno- mouse. type in the DKO mice compared with Srf Pf4-cKO mice is indepen- Comparison of ploidy between Mkl1 KO and DKO BM dent of the SRF cotranscriptional activities of MKL1 and MKL2. megakaryocytes indicates that DKO mice have a greater proportion From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

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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 Ͻ .05 between WT and DKO. (C) Venn diagrams showing genes with fold changes of 2 or more and FDR adjusted P value Ͻ .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.

of megakaryocytes in the 4N stage and a lower percentage with Ն Table 2. RNA sequencing cytoskeletal gene expression 8N DNA content. Recently, a relationship between MKL1 and FPKM guanine nucleotide exchange factors (GEFs) that control the final stages of cytokinesis was shown.33 GEF-H1 is a Rho-activating Gene WT SRF cKO MKL1 KO MKL2 cKO DKO protein that localizes to the contractile ring during normal mitosis. Actb 7795.17 2219.60* 2696.76* 5856.10 918.85* In normal differentiation, GEF-H1 expression decreases before the Cfl1 1002.17 620.35 548.28 690.11 723.96 first megakaryocyte endomitotic cycle and then increases as Cnn2 247.85 102.00* 42.33* 220.86 41.44* Flna 839.82 304.74* 309.33 583.87 56.52* maturation continues. Not only do Mkl1 KO megakaryocytes have Myh9 999.37 631.94 1017.94 1311.00 107.44* increased levels of GEF-H1, but Mkl1 KO megakaryocytes also do Pfn1 1935.96 918.24 1014.16 1359.71 1396.58 not down regulate GEF-H1 expression at any time during mega- Tpm2 1.04 1.02 0.54 1.03 4.08 karyocyte maturation. SRF binds at the GEF-H1 promoter in Tpm4 503.25 172.53* 259.49 510.30 53.64* hematopoietic cells, indicating a direct link between MKL1 and Vcl 201.99 65.07* 99.46 171.20 12.10* GEF-H1 regulation.34 RNA sequencing shows different expression levels of genes important in ECT2, another GEF, also plays a role in megakaryocyte maintaining and remodeling the actin cytoskeleton. Reads represent fragments per endomitosis. ECT2 concentrates in the midzone during cleavage kilobase per million and fold changes indicate log2 differences. Data are displayed in FPKM. furrow formation and is also required for the final stages of FPKM indicates fragments per kilobase of transcript per million; WT, wild type; cytokinesis. Disruption of ECT2 function leads to polyploidy in SRF, serum response factor; KO, knockout; cKO, conditional knockout; and DKO, HeLa cells.35 ECT2 is down-regulated in normal megakaryo- double knockout. *Differentially expressed genes reaching statistical significance (q Ͻ 0.05) com- cytes at the second endomitotic cycle (4N to 8N) and remains pared to WT. low as maturation proceeds. RNA sequencing of WT, Mkl1 KO, From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2326 SMITH et al BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11

Table 3. DKO megakaryocytes show deregulation of many Mkl2 Pf4-cKO, DKO, and Srf Pf4-cKO mature megakaryocytes megakaryocyte genes revealed higher expression of ECT2 in DKO and Srf Pf4-cKO FPKM samples. There was no significant difference in GEF-H1 expres- Gene WT SRF cKO MKL1 KO MKL2 cKO DKO sion between WT and Mkl1 KO mature megakaryocytes in these Aurkb 28.32 56.04 21.31 15.20 38.26 data sequencing data, which is in agreement with the initial Bcl2 2.05 1.58 2.42 2.13 0.91 report for megakaryocyte progenitors cultured for 3 days Bcl2l11 6.82 17.95* 12.75 6.67 4.71 in vitro. It may be that MKL1 and MKL2 differentially affect the Bcl2l1 177.03 75.50* 141.81 170.54 63.91* down-regulation of GEFs, which needs to occur to promote Cbfb 32.56 35.92 30.95 23.69 10.49* endomitosis. Future studies may be performed on megakaryo- Ccl5 14.29 81.43* 16.21 6.32 75.07* cytes from Mkl1 KO and DKO mice to assess the levels of Ccnd1 4.42 1.20 2.07 2.81 0.46 Ccnd3 539.47 319.20 448.96 547.66 429.51 critical GEFs that need to be down-regulated for the different Ccne1 5.36 7.25 4.45 2.90 9.88 stages of polyploidization and maturation. Ccne2 8.84 12.42 8.25 5.07 4.45 Analysis of differentially regulated genes and the abnormal Cd36 1.64 2.20 1.44 0.79 0.31 morphology of DKO megakaryocytes suggest that MKL1 and Cd9 1630.72 852.78 1449.95 1627.59 619.83 MKL2, through their interactions with SRF, control the complex Cdkn1a 346.36 255.60 328.10 292.71 228.05 architectural rearrangements that occur to allow proplatelet forma- Cxcl12 0.00 0.01 0.01 0.00 0.00 tion and, ultimately, platelet release. One of the genes that is Cxcl5 52.68 41.74 51.20 87.20 10.92* decreased in the DKO, the Srf Pf4-cKO, and the Srf Mx1-cKO is Cxcr4 11.60 104.87* 10.61 9.35 41.40* 14,15 Ets1 6.50 18.58* 9.49 8.98 3.80 Filamin A (Flna). The Flna Pf4-cKO was recently reported to 36 Etv6 83.30 73.60 85.93 109.23 13.31* have macrothrombocytopenia. This phenotype is consistent with F2r 538.79 403.48 510.62 754.55 73.00* the phenotype of DKO platelets and the Srf cKO platelets. Fli1 92.75 62.78 74.83 107.55 16.70* However, the Flna Pf4-cKO mice have structurally competent Gata1 136.72 61.85* 100.83 131.26 93.43 megakaryocytes, indicating the more pleiotropic nature of the Gata2 32.63 29.09 31.35 22.08 47.30 SRF/MKL deficiency. Future individual analyses of the major Gp1ba 225.28 101.01* 198.97 304.22 20.66* differentially expressed genes reported in the Srf Pf4-cKO microar- Gp5 270.36 99.97* 183.32 242.73 103.53 ray should allow a more comprehensive understanding of the many Gp6 120.38 68.16 119.21 170.59 41.46* Gp9 307.39 118.95* 222.33 264.54 212.19 ways in which the MRTF/SRF pathway contributes to megakaryo- Hsd3b1 0.19 0.04 0.13 0.08 0.01 cytopoiesis and ultimately to thrombopoiesis. Hsd3b2 0.01 0.01 0.00 0.01 0.00 One reason why DKO mice have fewer platelets than Srf Itga2 8.72 4.07 10.33 21.05* 0.08 Pf4-cKO mice may be the presence of residual SRF protein in Itga2b 2506.71 1231.66* 2305.33 2301.65 1637.24 megakaryocytes because the Pf4 promoter is not activated until Itga5 30.84 31.69 30.59 25.61 10.59 after megakaryocyte commitment occurs. Although a 90% de- Itgav 16.04 17.03 18.47 20.23 14.37 crease in SRF RNA was confirmed, assessment of SRF protein Itgb1 219.80 131.97 169.54 229.33 55.44* levels was not done.14 The Srf Mx-1 Cre mice, in which SRF is Itgb3 1285.89 667.85 1130.56 1556.85 243.91 Mafg 16.36 9.91 16.54 19.66 1.24 knocked out in all hematopoietic cells after administration of Mafk 25.26 15.14 21.01 19.13 6.49* Poly(I):Poly(C), have platelet counts more similar to our DKO 15 Mcl1 112.29 129.28 127.35 88.38 57.66 mice, supporting the residual protein hypothesis. Another possibil- Mpl 92.44 40.82* 57.96 116.22 23.74* ity is that the differences in phenotype between the DKO and Srf Myb 28.96 77.77* 41.61 18.42 33.15 Pf4-cKO mice may be attributable to SRF-independent functions Myh9 999.37 631.94 1017.94 1311.00 107.44* of the MRTFs. It has recently been shown that MKL1 can activate Nfe2 620.83 357.27 532.17 550.66 390.86 genes independently of its ability to bind SRF.37 P2rx1 135.55 63.86* 119.27 102.96 35.34* RNA quantification via deep sequencing revealed potential P2ry1 38.38 21.97 38.53 45.93 5.06* P2ry12 3.22 2.59 3.32 3.94 0.20 genes that could contribute to the phenotypic differences seen in Pf4 7444.13 4537.65 6066.01 6706.48 9408.82 DKO and Srf Pf4-cKO mice. Egfl7 and miR-126 are intriguing Ppbp 5550.82 589.32* 2275.74* 8246.70 482.21* candidates for further study. Egfl7 is highly expressed in endothe- Rab27b 572.79 262.99* 492.48 687.69 61.05* lial cells. The promoter region critical for its activation contains Rabggta 4.56 7.34 4.16 2.07 9.71 2 Ets-binding sites. Both Ets-1 and Ets-2 transactivate the Egfl7 Runx1 48.93 37.01 56.81 65.48 10.54* promoter in luciferase assays.38 These proteins are members of the Selp 682.35 210.07* 566.21 746.53 53.85* same superfamily as the ternary complex factors (TCFs), which Tal1 109.73 51.29* 94.37 106.55 17.59* compete with MRTFs for SRF binding.39 Tbxas1 470.61 202.36* 438.38 583.03 146.91* Tnfrsf1a 75.62 107.95 81.74 54.56 67.13 Expression of Egfl7 correlates with miR-126 expression Tubb1 119.49 85.87 107.73 183.81 12.97* whose function in megakaryocytopoiesis is unclear. Down- Vwf 893.73 420.56 727.50 1220.57 313.62 regulation of Spred1 by the increase in miR-126 expression Zfpm1 121.21 58.22 72.54 115.08 18.29* drives proliferation and activation of mast cells,40 functionally linking our observations of increased miR-126 expression with Megakaryocyte curated genes based on a literature review as being important in megakaryocyte differentiation23 show differential gene expression between the an increase in mast cell–related gene expression in the DKO different genotypes. Data are displayed in FPKM. megakaryocytes. Reports from zebrafish categorize c-Myb as a FPKM indicates fragments per kilobase of transcript per million; WT, wild type; direct target of miR-126. Morpholino knockdown of miR-126 SRF, serum response factor; KO, knockout; cKO, conditional knockout; and DKO, double knockout. results in increased c-Myb expression, which promotes red cell *Differentially expressed genes reaching statistical significance (q Ͻ 0.05) com- production at the expense of thrombocytes.41 Overexpression of pared to WT. miR-126 in human embryonic stem-cell derived CD34ϩ cells From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

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Table 4. DKO and Srf Pf4-cKO megakaryocytes have differential megakaryocyte transmembrane gene expression FPKM Gene WT SRF cKO MKL1 KO MKL2 cKO DKO

1110008F13Rik 168.39 108.66 124.53 118.02 162.05 Alox5ap 2324.05 1609.63 2435.91 2216.10 2811.82 Atp5g3 385.29 244.15 250.03 242.72 508.98 Bsg 319.57 233.30 278.88 310.46 513.28 Cd151 87.72 52.09 79.72 89.09 33.04* Cd47 30.43 31.40 25.75 23.65 0.00 Cd63 555.94 402.13 499.11 485.78 921.27 Cd9 1630.72 852.78 1449.95 1627.59 619.83 Cox4i1 521.67 401.89 414.34 408.12 1190.58 Cox8a 238.02 173.46 161.16 176.70 428.98 Cyba 155.52 323.36 134.54 87.42 765.77* Dhcr24 148.52 76.21 124.12 133.68 32.72* Dnajb4 42.37 25.26 36.92 47.77 11.62* Esam 126.13 68.79 80.62 146.31 91.92 Eya2 31.29 14.01* 41.15 24.88 10.51* Fads2 207.86 130.48 172.31 254.37 50.26* Fcer1g 542.87 519.26 403.81 409.80 682.78 Glipr1 245.40 205.21 238.57 161.53 227.67 Gp1ba 225.28 101.01* 198.97 304.22 20.66* Gp1bb 68.88 31.31 47.20 51.64 73.57 Gp49a 812.14 447.61 810.08 724.34 192.64* Gp5 270.36 99.97* 183.32 242.73 103.53 Gp9 307.39 118.95* 222.33 264.54 212.19 Gpr56 855.30 417.16 772.67 979.05 367.96 H2-K1 602.78 843.12 527.17 490.93 1433.99* Ifitm1 318.43 319.23 317.53 247.40 1536.24* Ifitm2 263.50 278.33 236.67 250.93 501.76 Ifitm3 580.84 578.90 410.03 463.05 1026.08 Itga2b 2506.71 1231.66* 2305.33 2301.65 1637.24 Itga6 665.79 277.35* 625.44 736.21 50.74* Itm2b 691.15 965.94 734.92 600.35 739.78 Laptm4a 138.76 107.84 125.00 133.70 102.47 Laptm5 2043.75 1786.38 2362.76 2407.72 512.81 Lilrb4 1168.14 626.80 1169.76 1092.51 273.49* Mcl1 112.29 129.28 127.35 88.38 57.66 Mpl 92.44 40.82* 57.96 116.22 23.74* Nrgn 437.18 203.67 256.78 303.27 226.77 P2rx1 135.55 63.86* 119.27 102.96 35.34* Pdzk1ip1 133.50 68.47 107.33 125.34 216.87 Ptpro 1.42 5.91 1.20 0.58 2.78 Rpn1 108.33 90.37 90.52 80.59 87.08 Scarb1 392.79 237.10 289.97 434.64 106.77* Scd2 205.74 244.30 199.91 229.47 84.20 Sec61a1 114.52 101.25 106.98 102.02 40.39 Selp 682.35 210.07* 566.21 746.53 53.85* Serinc3 523.01 476.27 552.93 434.22 177.98* Serpinb10-ps 176.75 79.74* 150.23 142.55 46.67* Serpinb2 1153.16 523.29* 1393.18 1015.87 98.99* Slc20a1 219.58 222.87 227.78 214.32 28.19* Slc25a3 413.05 291.79 320.12 306.51 428.44 Slc35d3 84.72 40.63 53.39 101.96 18.50* Slc37a2 2.20 6.28 1.83 0.80 1.74 Slc6a4 1717.84 542.43* 1667.10 1172.70 71.01* Ssr3 199.21 176.59 183.33 196.29 77.88 Tmbim4 840.92 338.18* 654.55 613.84 468.37 Tmem40 363.85 145.47* 267.51 294.62 264.04 Tmem66 133.46 97.40 117.65 115.44 108.09 Treml1 322.50 202.32 250.64 436.36 279.76

Data obtained from our RNA sequencing was compared to a list of the transmembrane proteins expressed speciﬁcally in megakaryocytes or up-regulated in megakaryocyte differentiation.24 Data are displayed in FPKM. FPKM indicates fragments per kilobase of transcript per million; WT, wild type; SRF, serum response factor; KO, knockout; cKO, conditional knockout; and DKO, double knockout. *Differentially expressed genes reaching statistical signiﬁcance (q Ͻ 0.05) compared to WT. From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2328 SMITH et al BLOOD, 13 SEPTEMBER 2012 ⅐ VOLUME 120, NUMBER 11

Table 5. Top genes increased in both DKO and Srf Pf4-cKO reported the aberrant expression of miR-126 in acute myeloid megakaryocytes leukemia-initiating cells.43-45 These reports suggest that deregu- FPKM lation of miR-126 and other miRs (eg, miR-155) may be the Gene WT SRF cKO MKL1 KO MKL2 cKO DKO additional hits that drive leukemic progression. This suggests an Camp 3.55 217.84 2.54 0.68 365.42 intriguing hypothesis in the case of t(1;22) AMKL; the fusion Cd177 0.62 25.74 0.49 0.29 19.06 protein OTT-MKL1 may disrupt the balance of TCFs and Cebpe 0.99 38.18 1.03 0.48 78.54 MRTFs at the Egfl7 promoter similar to the DKO mice. The Cldn15 2.83 50.78 3.05 1.93 51.11 result is the production of hyperproliferative cells that have Cma1 0.26 15.85 0.25 0.14 24.63 impaired megakaryocyte differentiation because of lack of Cpa3 2.95 46.38 4.18 2.93 53.55 normal MKL1 expression. This is also supported by competitive Ctsg 80.04 1110.46 77.54 42.09 1436.34 repopulation experiments in which overexpression of miR-126 Dmkn 4.20 30.77 4.87 2.36 73.51 provides an engraftment advantage.46 These findings reveal a Elane 126.51 2657.87 129.82 76.19 4523.18 connection between the MRTFs, Ets domain proteins, and Epx 1.39 16.91 0.79 1.00 21.83 Fcnb 2.37 196.80 4.08 1.98 107.95 miR-126 highlighting a pathway that may give insight into the G0s2 0.41 10.25 0.43 0.20 15.04 pathogenesis of AMKL. Gfi1 1.78 70.69 2.11 1.07 39.86 Gstm1 10.90 202.47 12.81 5.97 231.21 Lcn2 46.34 447.48 55.93 24.45 774.36 Acknowledgments Ltf 1.64 65.56 1.09 0.65 35.35 Mapk13 0.66 12.56 0.54 0.29 18.51 Mcpt8 7.46 141.38 10.37 6.24 362.91 The authors thank Dr Eric Olson for the Mkl2 floxed mice, Dr Mgst2 5.72 104.82 3.61 2.69 158.53 Stephen Morris for the Mkl1 KO mice, and Stephanie Donald- Mogat2 0.48 33.16 0.44 0.46 19.44 son for excellent mouse husbandry. The authors also thank Dr Ms4a3 33.51 348.80 38.93 19.18 483.45 Emanuela Bruscia, Alexandra Teixeira, Dr Betty Lawton, and Dr Ngp 9.93 764.69 5.76 2.56 834.41 Dennis Jones for thoughtful insights and careful editing of this Oscp1 0.74 10.77 0.75 0.74 13.66 manuscript. Pglyrp1 0.86 79.38 1.25 0.59 204.23 Prg2 19.58 220.00 19.20 14.11 858.80 This work was supported by National Institutes of Health (NIH) Prg3 2.13 17.73 1.91 1.44 71.50 grant F31 HL 094118 (to E.C.S.), and by NIH grants DK086267, Prss34 3.89 81.31 5.31 2.95 273.43 DK072442 (Yale Center of Excellence in Molecular Hematology), and Prss57 0.95 36.18 1.00 0.60 76.53 the Connecticut Stem Cell Fund (to D.S.K.), and HL106184 (to P.G.). Prtn3 123.47 1195.12 105.27 57.77 2720.61 Ramp1 1.50 15.73 1.72 0.75 22.26 S100a8 237.63 8015.96 226.82 68.84 9008.47 S100a9 137.93 7049.68 144.50 44.80 9372.56 Authorship Saa3 606.97 3825.61 628.93 244.01 9136.90 Slpi 30.75 244.71 36.52 17.42 663.24 Contribution: E.C.S. designed and performed experiments and Tst 1.56 15.88 1.14 1.26 35.82 wrote the manuscript; J.N.T., M.T.D., S.L., Y.G., and S.A.M. performed experiments and provided technical expertise; V.P.S. A list of genes more than 16-fold increased in DKO and Srf Pf4-cKO megakaryo- performed bioinformatics analysis; S.H., P.G., and J.E.I. contrib- cytes compared to WT megakaryocytes. Data are displayed in FPKM. FPKM indicates fragments per kilobase of transcript per million; WT, wild type; uted scientific knowledge; and D.S.K. provided mentorship and SRF, serum response factor; KO, knockout; cKO, conditional knockout; and DKO, intellectual input, and wrote the manuscript. double knockout. Conflict-of-interest disclosure: The authors declare no compet- ing financial interests. leads to a decrease in erythroid colony formation; megakaryo- Correspondence: Diane S. Krause, Department of Laboratory cyte potential was not assessed by this report.42 However, Medicine, Yale University School of Medicine, PO Box 208073, expression of miR-126 decreases with megakaryocyte differen- 333 Cedar St, New Haven, CT 06520-8073; e-mail: diane.krause@ tiation from adult CD34ϩ cells.43 Lastly, several groups have yale.edu. References

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2012 120: 2317-2329 doi:10.1182/blood-2012-04-420828 originally published online July 17, 2012

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

Elenoe C. Smith, Jonathan N. Thon, Matthew T. Devine, Sharon Lin, Vincent P. Schulz, Yanwen Guo, Stephanie A. Massaro, Stephanie Halene, Patrick Gallagher, Joseph E. Italiano Jr and Diane S. Krause

Updated information and services can be found at: http://www.bloodjournal.org/content/120/11/2317.full.html Articles on similar topics can be found in the following Blood collections Hematopoiesis and Stem Cells (3360 articles) Platelets and Thrombopoiesis (678 articles) Thrombocytopenia (215 articles)

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