Download MKL1 and MKL2 play redundant and crucial roles in

From www.bloodjournal.org by guest on August 3, 2017. 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 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 5ⴕ-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. (Blood. 2012;120(11):2317-2329)
Introduction
Biphenotypic megakaryocyte-erythroid precursors undergo
differentiation and endomitosis to become mature polyploid megakaryocytes that release platelets into the circulation. Despite
advances in our understanding of hematopoiesis, much remains
unknown regarding the regulation of megakaryocytopoiesis. Our
laboratory is focused on understanding the mechanisms of normal
megakaryocyte differentiation to better understand acute megakaryoblastic leukemia (AMKL). The reciprocal t(1;22) translocation that
is consistently associated with AMKL results in fusion of the
RBM15 (RNA-binding motif 15) and MKL1 (megakaryoblastic
leukemia 1) genetic loci.1,2 RBM15, an RNA-binding protein
whose function is not yet well defined, is differentially expressed in
hematopoiesis with the highest mRNA levels in progenitors and
lowest in differentiated blood cells.3 Here, we focus on the role of
the MKL1 family of proteins in megakaryocytopoiesis.
MKL1 (MRTF-A, MAL, BSAC) is a transcriptional cofactor
belonging to the myocardin-related transcription factor (MRTFs)
family that also includes myocardin and MKL2 (MRTF-B,
MAL16). These proteins function through association with
serum response factor (SRF), a ubiquitously expressed transcription factor.4 SRF is also activated by association with ternary
complex factors (Elk1, SAP1, SAP2).5 Transcriptional cofactor
binding determines the spatiotemporal activity of SRF. Srf
knockout (KO) mice die early in gestation because of abnormal
mesoderm development.6 Myocardin and Mkl2 KO mice are also
embryonic lethal because of abnormal cardiac development.7-9
Mkl1 KO mice show partial embryonic lethality, with some
embryos dying because of myocardial cell necrosis. However,
Mkl1 KO mice that complete gestation have normal lifespans.
Functions for MKL1 have been characterized in embryonic stem
cells, fibroblasts, smooth muscle cells, and neurons.10-12
Recently, we and others characterized the hematopoietic phenotypes of Mkl1 KO and megakaryocyte-specific Srf conditional KO
(Srf Pf4-cKO) mice.13-15 In each case, mice have thrombocytopenia
with increased numbers of immature megakaryocytes in the bone
marrow (BM) suggesting both a failure of normal megakaryocyte
maturation as well as abnormal formation of the megakaryocyte
and platelet cytoskeleton. Comparison of the Mkl1 KO and Srf
Pf4-cKO mice as well as in vitro cell-culture studies suggest that
the effect of MKL1 in megakaryocyte differentiation is mediated
by association with SRF.13 However, the hematopoietic phenotype
is far more severe in Srf Pf4-cKO than Mkl1 KO mice, which
indicates that other factors may act in conjunction with, or in the
absence of, MKL1 to promote SRF-mediated megakaryocyte
maturation. Possible contributing proteins include other members
of the MRTF family.
Here, we assessed the contribution of MKL2 to megakaryocytopoiesis, and found that while it is not required for effective
megakaryocyte maturation and platelet formation, MKL2 compensates for and mitigates the effects of MKL1 deficiency. Mice
lacking both MKL1 and MKL2 in the megakaryocyte lineage
have macrothrombocytopenia as well as platelet cytoskeletal
abnormalities and severely impaired platelet activation. In addition
to the peripheral blood defects, there is abnormal megakaryocyte
Submitted March 30, 2012; accepted July 6, 2012. Prepublished online as
Blood First Edition paper, July 17, 2012; DOI 10.1182/blood-2012-04-420828.
The publication costs of this article were defrayed in part by page charge
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 䡠 VOLUME 120, NUMBER 11
2317
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2318
BLOOD, 13 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 11
SMITH et al
ultrastructure. Surprisingly, the phenotype of double KO (DKO)
mice is more extreme than that of the Srf Pf4-cKO mice, strongly
suggesting that MRTFs function in ways that are independent of
their SRF transcriptional coactivation activities.
previously described.20 Samples were examined with an Axiovert 200
microscope (Carl Zeiss Inc) equipped with a 63⫻ NA 1.4 oil-immersion
objective. Images were obtained using a charge-coupled device camera
(Hamamatsu Photonics) and phalloidin quantified using NIH ImageJ
software.
Methods
DNA and RNA analysis
Mouse strains
All procedures were performed in compliance with relevant laws and
institutional guidelines and were approved by the Yale University Institutional Animal Care and Use Committee. Mkl2F/F mice16 were crossed with
platelet factor 4 (Pf4) Cre17-expressing mice. These mice were then bred
onto the Mkl1 KO background.7 Mkl1 KO and Pf4-Cre mice crossed with
SrfF/F were as previously described.13,14 All mice were on a C57Bl/6J
background.
Flow cytometry, cell sorting, and in vitro culture
To determine mouse BM progenitor populations, freshly isolated BM was
incubated with anti-CD16/CD32 antibodies (Fc block; BD Biosciences),
stained with PE-biotin lineage detection cocktail (Miltenyi Biotec),
allophycocyanin-H7 CD117/c-kit (BD Biosciences), Alexa 647 Sca-1,
PE-Cy5 CD150, PE-Cy7 CD105 (BioLegend), and FITC CD41 (BD
Biosciences), and analyzed (LSRII; BD Biosciences) or sorted (FACS Aria,
BD Biosciences; or MoFlo, Beckman Coulter) as previously described.18
For sorting experiments, BM was first enriched for stem/progenitor cells
using immunomagnetic separation (BD Biosciences). LSK (lineage [lin]
negative, CD117 positive, Sca-1 positive), PreMegE (lin negative, CD117
positive, CD41 negative, CD150 positive, CD105 negative), and MkP (lin
negative, CD117 positive, CD41 positive) were assayed. For DNA content
analysis, unfractionated whole BM was stained with FITC CD41, then
treated with 70% ethanol overnight followed by digestion with 20 ␮g/mL
RNase (Sigma-Aldrich) on ice for 4 hours. Cells were resuspended with
10 ␮g/mL propidium iodide (Sigma-Aldrich) before analysis on a FACSCalibur (BD Biosciences). Platelet activation was assessed using FITC CD41/61
and PE JON/A (Emfret Analytics). Flow cytometric data were analyzed
using FlowJo software (TreeStar). PreMegE and MkP were differentiated
for 5 or 3 days, respectively, in megakaryocyte differentiation medium
containing StemSpan Serum Free Expansion Media (StemCell Technologies) supplemented with 30% BIT 9500, L-glutamine (Life Technologies),
penicillin/streptomycin (P/S; Life Technologies), and 50 ng/mL murine
thrombopoietin (mTPO; ConnStem). Fetal liver megakaryocytes were
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.
Platelet preparations
Peripheral blood was collected from the retro-orbital sinus into tubes
containing acid citrate dextrose (ACD) anticoagulant. Platelet-rich plasma
was prepared as previously described.19 For flow cytometry, 50 ␮L of
whole blood was added to 200 ␮L of 20 U/mL heparin in Tris-buffered
saline (20mM Tris-HCl, 137mM NaCl). After further dilution with 1 mL of
2mM CaCl2 in modified Tyrode-HEPES buffer (5mM HEPES, 140mM
NaCl, 2.7mM KCl, 5.5mM dextrose, 0.42mM Na2HPO4, 12mM NaHCO3),
platelets were stimulated with 1mM adenosine 5⬘-diphosphate (ADP;
Sigma-Aldrich).
DNA and RNA were extracted using the QIAGEN DNeasy Blood and
Tissue and Ambion RNAqueous Micro kits, respectively. cDNA was made
using Superscript III (Life Technologies) with random primers (Life
Technologies). Quantification was performed using a CFX96 C1000
thermal cycler (Bio-Rad) using TaqMan gene expression assays (Applied
Biosystems–Life Technologies): murine epidermal growth factor-like domain 7 (EGFL7) and murine Spred1, and eukaryotic 18S as an internal
control. U6 RNA was used as an internal control for miR-126 as assayed by
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
were collected from at least 8 different mice for each genotype and pooled
for RNA sequencing. Library preparation and sequencing were performed
by the Yale Stem Cell Genomics Core Facility using the Illumina TruSeq
RNA Sample Preparation kit. Samples were sequenced on an Illumina
HiSeq 2000 using 50-cycle single-end sequencing. FASTQ format sequencing reads were aligned to the mm9 genome using Tophat Version 1.3.1
software.21 The cufflinks, cuffmerge, and cuffdiff Version 1.3.0 programs
were used to identify differences in Ensembl transcripts.22 The analysis
used upper-quartile normalization, multiread, and GC fragment bias
corrections, and masking of reads in rRNA and tRNA genes. Sample
comparisons are displayed using the R heatmap.2, lumi, and VennDiagram
packages. Data are publicly available through Gene Expression Omnibus
(file numbers pending). Statistically significant differences between genotypes were defined by genes having more than 10 fragments per kilobase of
transcript per million (FPKM) reads in at least 1 of the 2 samples compared
and a q value of less than 0.05.
Bone marrow histology
Femurs were fixed overnight in 4% paraformaldehyde, decalcified in
Decalcifier I solution (Surgipath) overnight, and transferred to 70% ethanol
for processing by the Research Histology Facility at Yale School of
Medicine for 5-␮m longitudinal paraffin sections, hematoxylin and eosin
(H&E) staining, and immunohistochemistry for von Willebrand factor
(anti-VWF; DAKO).
Bleeding time measurements
Three-week-old mice were anesthetized using isoflurane (Butler Animal
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
bleeding stopped.
Statistical analysis
Statistical significance was assessed using the 2-tailed unpaired t test with
Prism software (GraphPad).
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
immediately fixed with 2% paraformaldehyde or allowed to spread for
20 minutes and then fixed (platelets only). Permeabilization, blocking,
and staining with using a ␤1 tubulin antibody (Genemed Synthesis Inc)
and Alexa Fluor 568 Phalloidin (Life Technologies) were conducted as
The hematopoietic phenotype of megakaryocyte-specific (Pf4-Cre)
Srf conditional KO (Srf Pf4-cKO) is more severe than that of Mkl1
KO mice,14 suggesting compensation or redundancy in Mkl1 KO
mice. Because there are multiple transcriptional coactivators of
SRF, multiple other proteins may mitigate the effects of MKL1
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 13 SEPTEMBER 2012 䡠 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
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 Mkl2F/F mice. Mkl2F/F mice
without Pf4-Cre were negative controls.
deficiency. Possible genes that could compensate for the loss of
MKL1 are Myocardin and Mkl2. Myocardin’s expression is
restricted to the heart. We investigated whether MKL2, known to
be expressed in multiple cell types, is expressed in megakaryocytes. RNA was taken from PreMegE cells sorted by flow
cytometry immediately postsort (day 0) and after culturing 5 days
in megakaryocyte differentiation media. MKL2 expression increased during megakaryocyte differentiation, although not as
dramatically as MKL1 (Figure 1A). We hypothesized that Mkl2
mRNA would be up-regulated to compensate for the lack of MKL1
in the Mkl1 KO mice. However, there was no increase in Mkl2
expression in megakaryocytes derived from PreMegE cells in Mkl1
KO mice compared with WT controls (Figure 1B).
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 ⬍ .0001) decreased
(187 000 ⫾ 56 000 platelets/␮L) compared with WT
(730 000 ⫾ 148 000 platelets/␮L), Mkl1 KO (460 000 ⫾ 94 000
platelets/␮L), and Mkl2 Pf4-cKO (715 000 ⫾ 120 000 platelets/
␮L; Figure 2A). In the absence of MKL2, 1 copy of MKL1 is
sufficient to maintain normal platelet counts (643 000 ⫾ 157 000
platelets/␮L). In contrast, mice with 1 copy of MKL2 in the
absence of MKL1 have platelet counts (325 000 ⫾ 54 000 platelets/
␮L) significantly higher than DKO mice, but lower than WT mice.
Srf Pf4-cKO platelet numbers (416 000 ⫾ 74 000 platelets/␮L)
were decreased compared with WT, consistent with previous
reports,14 however, they were significantly higher than DKO mice.
DKO mice also had increased mean platelet volume (MPV;
Figure 2B) as assessed by peripheral blood smears (Figure 2C).
Further characterization of the DKO animals indicated defective hemostasis. Bleeding times of the DKO mice (171 ⫾ 86 seconds) were significantly increased over wild-type (WT; 35 ⫾ 14 seconds), Mkl1 KO (73 ⫾ 52 seconds), and Mkl2 Pf4-cKO
(34 ⫾ 13 seconds) mice (Figure 2D). It is not clear why 3 of
13 DKO mice had bleeding times within the normal range. The
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
state, WT, Mkl1 KO, Mkl2 Pf4-cKO, and Srf Pf4-cKO platelets
displayed normal forward scatter (FSC) and side scatter (SSC). In
contrast, DKO platelet morphology was less uniform and the
platelet and red cell populations were not well separated (Figure 2E, Table 1), consistent with increased MPV. After ADP
treatment, there was an increase in activated CD41/61 heterodimer
on the surface of WT, Mkl1 KO, and Mkl2 Pf4-cKO platelets using
a PE-conjugated JON/A antibody (Figure 2F, Table 1). In contrast,
DKO platelets did not respond to ADP; there was no conformational change of the CD41/61 heterodimer to allow for JON/A
antibody binding. In addition, DKO platelets did not display any
shape change within the FSC/SSC gate after ADP treatment.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2320
SMITH et al
BLOOD, 13 SEPTEMBER 2012 䡠 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 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.
DKO platelets have cytoskeleton abnormalities and atypical
morphology
Flow cytometry suggested altered platelet structure and shape.
Because many cytoskeleton genes are SRF targets, organization of
polymerized actin and microtubules of the cytoskeleton were
assessed. Immunofluorescence images of platelets, both resting and
spread, showed differences in the actin and microtubule cytoskel-
eton organization in DKO platelets as assayed by phalloidin and
␤1-tubulin, a megakaryocyte lineage-specific isoform of tubulin
(Figure 3A). Resting WT and Mkl2 Pf4-cKO platelets showed
significant filamentous actin staining (shown in red) and distinct
microtubule rings (shown in green) characteristic of platelets
(Figure 3Ai,iii). Mkl1 KO platelets had an organized microtubule
ring but lacked phalloidin staining (Figure 3Aii). After contact with
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 13 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 11
MKL2 IN MEGAKARYOCYTOPOIESIS
2321
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 ⫾ .32
25 ⫾ .84
29 ⫾ .42
47 ⫾ 3.9*
30 ⫾ .53
29 ⫾ .46
33 ⫾ 1.2
52 ⫾ 4.9*
JON/A, MFI
17 ⫾ .4
16 ⫾ .26
17 ⫾ .25
20 ⫾ 1.0*
106 ⫾ 16
104 ⫾ 11
108 ⫾ 48
30 ⫾ 1.4*
Mean FSC and MFI of JON/A staining was averaged and the SD displayed (n ⫽ 4).
ADP indicates adenosine 5⬘-diphosphate; WT, wild type; KO, knockout; cKO, conditional knockout; DKO, double knockout; FSC, forward scatter; and MFI, mean
fluorescence intensity.
*P ⬍ .001.
glass, WT and Mkl2 Pf4-cKO platelets retract their tubulin coils,
and polymerize actin to extend filopodia and lamellopodia, ultimately causing them to spread (Figure 3Av,vii). Although spread
Mkl1 KO platelets had some microtubule reorganization, most
retained partial microtubule coil morphology and the actin cytoskeleton remained disorganized (Figure 3Avi). DKO platelets did not
change shape after adhesion to coverslips and had no filamentous
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 ⬍ .001).
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2322
SMITH et al
activation, DKO platelets were often amorphously shaped, unlike
the characteristic discoid shape of WT platelets. Quantification of
phalloidin by immunofluorescence confirmed the decrease in
polymerized actin (Figure 3A bottom panels). Of the DKO platelets with detectable ␤1 tubulin, the marginal tubulin coil was
thicker than their WT and single KO counterparts and revealed
distinct heterogeneity in size and shape (Figure 3Aiv). The bright
␤1-tubulin immunofluorescence signal in the DKO platelets also
suggested an increase in ␤1-tubulin protein.
As suggested by flow cytometry (Figure 2E), DKO platelets
have less uniform granularity and size than WT, Mkl1 KO, and
Mkl2 Pf4-cKO platelets. To analyze this defect in more detail,
platelet ultrastructure was assessed by electron microscopy. The
images shown highlight the defective cytoskeleton and abnormal
granular contents of the DKO platelets (Figure 3B). A unique
feature of platelets is the microtubule ring that lies just below the
platelet surface. This ring is a single microtubule, looped 8-12 times,
allowing a 100-micron tubule to exist within a 2- to 4-micron
platelet, giving platelets their characteristic discoid shape. Electron
micrograph analysis of WT, Mkl1 KO, and Mkl2 Pf4-cKO platelets
revealed normal numbers of microtubule rings in cross-section8-12
at their tips while DKO platelets were seen to have more (up to 17)
microtubules when cross-sectioned (Figure 3B thick black arrows).
These data correlate with the increased intensity of the ␤1-tubulin
immunostaining (Figure 3Aiv,viii). In general, the DKO platelets
were amorphously shaped with poorly defined borders, unlike the
uniformly discoid shape of the WT platelets. Most notable,
however, was the loss of heterogeneity of the granular contents of
the DKO platelets. Some of the DKO platelets had granular
distribution very similar to WT, while others lacked both ␣ and
dense granules.
DKO BM has an accumulation of immature megakaryocytes
Platelet defects in peripheral blood suggest abnormal BM megakaryocytopoiesis. Immunohistochemistry for VWF on BM sections
(Figure 4A) revealed an increase in megakaryocyte number in
DKO, and, to a lesser extent, Mkl1 KO mice (already reported),
compared with WT and Mkl2 Pf4-cKO BM. The VWF-positive
megakaryocytes in the DKO were often small, with low ploidy and
irregular cell morphology, consistent with the staining patterns
found in Srf Pf4-cKO mice. H&E staining did not reveal any
differences in BM cellularity (Figure 4B).
Flow cytometric analysis of DKO BM CD41⫹ cells showed a
statistically significant (P ⬍ .01) shift to lower ploidy megakaryocytes compared with WT (Figure 4C-D). Ploidy measures the
DNA content of cells, thus assessing the number of endomitotic
cycles a cell has undergone. Mkl1 KO and DKO BM had a
similar percentage of megakaryocytes that were 2N. However,
there were fewer DKO megakaryocytes with high ploidy (ⱖ 8N)
compared with Mkl1 KO, resulting in a statistically significant
increase in 4N cells in DKO compared with Mkl1 KO mice. This
suggests that MKL2 may play a role in promoting the second
and subsequent endomitotic cycles as opposed to the first 2N to
4N endomitosis. There was a statistically significant decrease in
mean ploidy of DKO CD41⫹ cells (n ⫽ 4) compared with WT
(n ⫽ 4; P ⬍ .001) and Mkl1 KO (n ⫽ 4; P ⬍ .05) megakaryocytes. Consistent with the lack of a platelet phenotype, Mkl2
Pf4-cKO CD41⫹ BM cells did not show any difference from WT
BM. The increased number of DKO BM megakaryocytes was
confirmed by flow cytometry (Figure 4E); DKO marrow also had
greater percentages of PreMegE and MkP versus WT (Figure 4F).
As shown before,13,15 Mkl1 KO mice had a statistically significant
BLOOD, 13 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 11
(P ⬍ .05) increase over WT cells only in the MkP and total CD41⫹
populations. DKO megakaryocytes have a statistically significant
increase in 4N megakaryocytes at the expense of ⱖ 8N megakaryocytes compared with Mkl1 KO mice; this suggests that DKO
mice have more impaired megakaryocyte maturation than Mkl1
KO mice.
Megakaryocytes from DKO mice have abnormal cytoplasmic
ultrastructure
Ultrastructural analysis revealed morphologic abnormalities in
DKO megakaryocytes (Figure 5A). Compared with WT, DKO
megakaryocytes have cytoplasmic regions that are devoid of
demarcation membrane and increased numbers of vacuoles, which
normally appear during later stages of maturation (Figure 5Aiii,iv).
In addition, the areas of microvesiculation that occur around the
plasma membrane of WT megakaryocytes were present throughout
the cytoplasm of DKO megakaryocytes in the process of proplatelet formation and may account for the heterogeneity in platelet
shape, size, and granule content observed in these mice
(Figure 5Aiii,iv). Analysis of Srf Pf4-cKO mice suggests that
defects in the actin cytoskeleton are responsible for the abnormal
megakaryocyte and platelet morphology.14 Measurement of the
filamentous actin was determined by quantitative immunofluorescence microscopy of phalloidin (Figure 5B), which showed a
significant decrease in actin organization in the DKO megakaryocytes, consistent with the hypothesis that the actin cytoskeleton is
disordered.
RNA sequencing reveals SRF-independent functions for
MRTFs in megakaryocyte differentiation
Investigation of the role of MKL2 in megakaryocytopoiesis
suggests that MKL2 functions in the absence of MKL1 to promote
megakaryocyte maturation, and that much of this effect is mediated
via activation of SRF target genes. However, while the data
indicate that many of the defects in DKO mice phenocopy those of
SRF cKO mice, the platelet counts of DKO mice are more
dramatically reduced. This may be because of SRF-independent
functions of the MRTFs. To assess these novel functions, we
sequenced the transcriptomes of WT, Mkl1 KO, Mkl2 Pf4-cKO,
DKO, and Srf Pf4-cKO MkPs differentiated in vitro for 3 days with
TPO. We confirmed that the population of differentiated megakaryocytes analyzed from each genotype consisted of a relatively pure
(⬎ 93%) CD41⫹/CD42⫹ cell population without myeloid contamination (data not shown).
Analysis of the deep sequencing data revealed that DKO
megakaryocytes had the most altered expression profile compared
with the other 4 genotypes. A majority of the pairwise comparisons
did not have many genes with greater than 2-fold differential gene
expression (supplemental Figure 1, available on the Blood Web
site; see the Supplemental Materials link at the top of the online
article). A total of 969 genes showed a ⬎ 2-fold difference between
WT and Srf Pf4-cKO, while 2906 genes had a ⬎ 2-fold difference
between WT and DKO megakaryocytes. Between Srf Pf4-cKO and
DKO megakaryocytes, there were 3298 genes with a ⬎ 2-fold
change in expression (Figure 6A-B, supplemental Figure 1). As
expected, the DKO megakaryocytes significantly differed from
Mkl1 KO megakaryocytes and consistent with the more extreme
phenotype of the DKO mice, DKO megakaryocyte gene expression
was more dissimilar to WT than any other genotype (Figure 6A,
supplemental Figure 1). Although there were many genes that
showed similar changes in gene expression in WT compared with
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 13 SEPTEMBER 2012 䡠 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 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).
DKO and Srf Pf4-cKO, most genes were DKO-specific (Figure 6B).
The appearance of DKO-specific gene expression differences is
unlikely because of incomplete removal of SRF in Srf Pf4-cKO,
since there are also many genes that differ between Srf Pf4-cKO
and WT, but not between DKO and WT (Figure 6B and data not
shown). As expected, genes involved in cytoskeletal organization
were significantly decreased in Mkl1 KO, DKO, and Srf Pf4-cKO
megakaryocytes compared with WT (Table 2).
The differentially expressed genes open several interesting
avenues of research that give insight into the cellular properties
that drive the dramatic DKO platelet phenotype. We mined the
RNA sequencing data for genes already defined by others as
megakaryocyte-specific and genes highly expressed during
megakaryocyte differentiation.23,24 Of the 56 genes curated by
the Papoutsakis group as being megakaryocyte related by
literature review, 24 were deregulated by loss of both MKL1 and
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2324
SMITH et al
BLOOD, 13 SEPTEMBER 2012 䡠 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 ⬍ .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
megakaryocytes, 22 had altered gene expression in DKO
megakaryocytes and 14 were deregulated in SRF Pf4-cKO
megakaryocytes, while none of the 58 genes was significantly
affected by loss of MKL1 alone (Table 4). Slc35d3, one of the
genes that is significantly decreased uniquely in DKO cells, is
essential for platelet dense granule biogenesis.25,26 Of the
35 genes that were up-regulated at least 16-fold compared with
WT in both the Srf cKO and DKO (Table 5), 20 are strongly
associated with defense response, particularly neutrophil and
mast cell differentiation (for example, C/EBP epsilon). These
data suggest that SRF, via activation by MRTFs, may act as a
master switch to turn off myeloid differentiation programs
during megakaryocyte commitment.
Epidermal growth factor-like domain 7 (Egfl7) was the most
significantly overexpressed gene in DKO compared with WT,
Mkl1 KO, Mkl2 Pf4-cKO, and Srf Pf4-cKO megakaryocytes. Its
expression was up-regulated 10-fold in DKO megakaryocytes by
RNA sequencing analysis and greater than 6-fold by quantitative
PCR analysis (Figure 6C and data not shown). Egfl7 protein is
expressed and secreted by endothelial cells, and Egfl7 knockdown in primary human endothelial cells results in decreased
proliferation and migration.27 Within Egfl7’s seventh intron is
miR-126. Consistent with the Egfl7 data, expression of miR-126
as determined by quantitative PCR is also significantly increased
in DKO megakaryocytes (Figure 6C). Gene set enrichment
analysis28,29 confirmed the decrease in expression of miR-126
target genes in DKO megakaryocytes compared with WT megakaryocytes (supplemental Figure 2). RNA sequencing and qPCR
of DKO megakaryocytes showed decreased expression of Spred1,
a published target of miR-12630 (Figure 6C and data not shown).
Therefore, the more extreme megakaryocyte and platelet phenotype in the DKO mice compared with Srf Pf4-cKO mice is independent of the SRF cotranscriptional activities of MKL1 and MKL2.
Discussion
Megakaryocytes lacking expression of MKL1 and MKL2 have
both defective megakaryocytopoiesis and thrombopoiesis. Mkl1
KO mice with megakaryocyte-specific loss of MKL2 have increased megakaryocyte progenitors and immature megakaryocytes
in the BM. DKO mice have macrothrombocytopenia and increased
bleeding times as a result of ineffective megakaryocytopoiesis and
defects in platelet formation and activation. Many, but not all, of
the differences between the Mkl1 KO and the SRF Pf4-cKO can be
explained by the presence of MKL2.
This study is the first to describe a role for MKL2 in
hematopoiesis. Although MKL2 was known to function in the brain
and smooth muscle cells,8,16,31 investigators had dismissed MKL2
as a mediator of megakaryocytopoiesis because expression in
human megakaryocytes is low.32 While our mouse studies also
show that MKL2 is not as highly expressed as MKL1,
megakaryocyte-specific deletion of MKL2 in the absence of MKL1
indicates that MKL2 plays a role, albeit redundant, in megakaryocytopoiesis. SRF target genes that are differentially expressed in
the absence of SRF, MKL1, or MKL2 have been determined by this
study and others.14,15 Future studies will be focused on determining
how these differentially expressed target genes cause the differences in phenotypes between the DKO and Srf Pf4-cKO megakaryocytes and platelets. To further determine the mechanisms by which
MKL1 and MKL2 act to promote megakaryocytopoiesis, it will be
essential to know where SRF, MKL1, and MKL2 are bound on the
chromatin during megakaryocyte differentiation. Furthermore, it
will be important to determine whether the ability of MKL2 to
compensate for MKL1 is because of MKL2 occupation of the
genomic sites left vacant by the MKL1 deficiency in an Mkl1 KO
mouse.
Comparison of ploidy between Mkl1 KO and DKO BM
megakaryocytes indicates that DKO mice have a greater proportion
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 13 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 11
MKL2 IN MEGAKARYOCYTOPOIESIS
2325
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.
Table 2. RNA sequencing cytoskeletal gene expression
FPKM
Gene
WT
SRF cKO
MKL1 KO
MKL2 cKO
DKO
Actb
7795.17
2219.60*
2696.76*
5856.10
918.85*
Cfl1
548.28
690.11
723.96
1002.17
620.35
Cnn2
247.85
102.00*
Flna
839.82
304.74*
309.33
583.87
56.52*
Myh9
999.37
631.94
1017.94
1311.00
107.44*
Pfn1
42.33*
220.86
41.44*
1935.96
918.24
1014.16
1359.71
Tpm2
1.04
1.02
0.54
1.03
1396.58
Tpm4
503.25
172.53*
259.49
510.30
53.64*
Vcl
201.99
65.07*
99.46
171.20
12.10*
4.08
RNA sequencing shows different expression levels of genes important in
maintaining and remodeling the actin cytoskeleton. Reads represent fragments per
kilobase per million and fold changes indicate log2 differences. 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 ⬍ 0.05) compared to WT.
of megakaryocytes in the 4N stage and a lower percentage with
ⱖ 8N DNA content. Recently, a relationship between MKL1 and
guanine nucleotide exchange factors (GEFs) that control the final
stages of cytokinesis was shown.33 GEF-H1 is a Rho-activating
protein that localizes to the contractile ring during normal mitosis.
In normal differentiation, GEF-H1 expression decreases before the
first megakaryocyte endomitotic cycle and then increases as
maturation continues. Not only do Mkl1 KO megakaryocytes have
increased levels of GEF-H1, but Mkl1 KO megakaryocytes also do
not down regulate GEF-H1 expression at any time during megakaryocyte maturation. SRF binds at the GEF-H1 promoter in
hematopoietic cells, indicating a direct link between MKL1 and
GEF-H1 regulation.34
ECT2, another GEF, also plays a role in megakaryocyte
endomitosis. ECT2 concentrates in the midzone during cleavage
furrow formation and is also required for the final stages of
cytokinesis. Disruption of ECT2 function leads to polyploidy in
HeLa cells.35 ECT2 is down-regulated in normal megakaryocytes at the second endomitotic cycle (4N to 8N) and remains
low as maturation proceeds. RNA sequencing of WT, Mkl1 KO,
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2326
BLOOD, 13 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 11
SMITH et al
Table 3. DKO megakaryocytes show deregulation of many
megakaryocyte genes
FPKM
Gene
WT
Aurkb
28.32
56.04
21.31
15.20
38.26
Bcl2
2.05
1.58
2.42
2.13
0.91
Bcl2l11
6.82
17.95*
12.75
6.67
177.03
75.50*
141.81
170.54
Cbfb
32.56
35.92
30.95
23.69
10.49*
Ccl5
14.29
81.43*
16.21
6.32
75.07*
Bcl2l1
SRF cKO
MKL1 KO
MKL2 cKO
DKO
4.71
63.91*
Ccnd1
4.42
1.20
2.07
2.81
0.46
Ccnd3
539.47
319.20
448.96
547.66
429.51
Ccne1
5.36
7.25
4.45
2.90
9.88
Ccne2
8.84
12.42
8.25
5.07
4.45
Cd36
1.64
2.20
1.44
0.79
0.31
1630.72
852.78
1449.95
1627.59
619.83
228.05
Cd9
Cdkn1a
346.36
255.60
328.10
292.71
Cxcl12
0.00
0.01
0.01
0.00
0.00
Cxcl5
52.68
41.74
51.20
87.20
10.92*
Cxcr4
41.40*
11.60
104.87*
10.61
9.35
Ets1
6.50
18.58*
9.49
8.98
Etv6
83.30
73.60
85.93
109.23
13.31*
F2r
538.79
403.48
510.62
754.55
73.00*
Fli1
92.75
62.78
74.83
107.55
16.70*
136.72
61.85*
100.83
131.26
93.43
Gata1
3.80
Gata2
32.63
29.09
31.35
22.08
47.30
Gp1ba
225.28
101.01*
198.97
304.22
20.66*
Gp5
270.36
99.97*
183.32
242.73
Gp6
120.38
68.16
119.21
170.59
Gp9
307.39
118.95*
222.33
264.54
212.19
0.01
103.53
41.46*
Hsd3b1
0.19
0.04
0.13
0.08
Hsd3b2
0.01
0.01
0.00
0.01
0.00
Itga2
8.72
4.07
10.33
21.05*
0.08
Itga2b
2305.33
2301.65
1637.24
Itga5
30.84
31.69
30.59
25.61
10.59
Itgav
16.04
17.03
18.47
20.23
14.37
Itgb1
219.80
131.97
169.54
229.33
55.44*
Itgb3
1285.89
667.85
1130.56
1556.85
243.91
Mafg
16.36
9.91
16.54
19.66
1.24
Mafk
25.26
15.14
21.01
19.13
Mcl1
112.29
129.28
127.35
88.38
57.66
Mpl
92.44
40.82*
57.96
116.22
23.74*
Myb
28.96
77.77*
41.61
18.42
Myh9
999.37
631.94
1017.94
1311.00
107.44*
Nfe2
620.83
357.27
532.17
550.66
390.86
P2rx1
135.55
63.86*
119.27
102.96
P2ry1
38.38
21.97
38.53
45.93
5.06*
P2ry12
3.22
2.59
3.32
3.94
0.20
Pf4
7444.13
4537.65
6066.01
6706.48
9408.82
Ppbp
5550.82
589.32*
2275.74*
8246.70
482.21*
Rab27b
572.79
262.99*
492.48
687.69
61.05*
Rabggta
4.56
7.34
4.16
2.07
9.71
48.93
37.01
56.81
65.48
10.54*
Selp
682.35
210.07*
566.21
746.53
53.85*
Tal1
109.73
51.29*
94.37
106.55
17.59*
Tbxas1
470.61
202.36*
438.38
583.03
146.91*
Runx1
Tnfrsf1a
2506.71
1231.66*
6.49*
33.15
35.34*
75.62
107.95
81.74
54.56
67.13
Tubb1
119.49
85.87
107.73
183.81
12.97*
Vwf
893.73
420.56
727.50
1220.57
Zfpm1
121.21
58.22
72.54
115.08
313.62
18.29*
Megakaryocyte curated genes based on a literature review as being important in
megakaryocyte differentiation23 show differential gene expression between the
different genotypes. 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 ⬍ 0.05) compared to WT.
Mkl2 Pf4-cKO, DKO, and Srf Pf4-cKO mature megakaryocytes
revealed higher expression of ECT2 in DKO and Srf Pf4-cKO
samples. There was no significant difference in GEF-H1 expression between WT and Mkl1 KO mature megakaryocytes in these
data sequencing data, which is in agreement with the initial
report for megakaryocyte progenitors cultured for 3 days
in vitro. It may be that MKL1 and MKL2 differentially affect the
down-regulation of GEFs, which needs to occur to promote
endomitosis. Future studies may be performed on megakaryocytes from Mkl1 KO and DKO mice to assess the levels of
critical GEFs that need to be down-regulated for the different
stages of polyploidization and maturation.
Analysis of differentially regulated genes and the abnormal
morphology of DKO megakaryocytes suggest that MKL1 and
MKL2, through their interactions with SRF, control the complex
architectural rearrangements that occur to allow proplatelet formation and, ultimately, platelet release. One of the genes that is
decreased in the DKO, the Srf Pf4-cKO, and the Srf Mx1-cKO is
Filamin A (Flna).14,15 The Flna Pf4-cKO was recently reported to
have macrothrombocytopenia.36 This phenotype is consistent with
the phenotype of DKO platelets and the Srf cKO platelets.
However, the Flna Pf4-cKO mice have structurally competent
megakaryocytes, indicating the more pleiotropic nature of the
SRF/MKL deficiency. Future individual analyses of the major
differentially expressed genes reported in the Srf Pf4-cKO microarray should allow a more comprehensive understanding of the many
ways in which the MRTF/SRF pathway contributes to megakaryocytopoiesis and ultimately to thrombopoiesis.
One reason why DKO mice have fewer platelets than Srf
Pf4-cKO mice may be the presence of residual SRF protein in
megakaryocytes because the Pf4 promoter is not activated until
after megakaryocyte commitment occurs. Although a 90% decrease in SRF RNA was confirmed, assessment of SRF protein
levels was not done.14 The Srf Mx-1 Cre mice, in which SRF is
knocked out in all hematopoietic cells after administration of
Poly(I):Poly(C), have platelet counts more similar to our DKO
mice, supporting the residual protein hypothesis.15 Another possibility is that the differences in phenotype between the DKO and Srf
Pf4-cKO mice may be attributable to SRF-independent functions
of the MRTFs. It has recently been shown that MKL1 can activate
genes independently of its ability to bind SRF.37
RNA quantification via deep sequencing revealed potential
genes that could contribute to the phenotypic differences seen in
DKO and Srf Pf4-cKO mice. Egfl7 and miR-126 are intriguing
candidates for further study. Egfl7 is highly expressed in endothelial cells. The promoter region critical for its activation contains
2 Ets-binding sites. Both Ets-1 and Ets-2 transactivate the Egfl7
promoter in luciferase assays.38 These proteins are members of the
same superfamily as the ternary complex factors (TCFs), which
compete with MRTFs for SRF binding.39
Expression of Egfl7 correlates with miR-126 expression
whose function in megakaryocytopoiesis is unclear. Downregulation of Spred1 by the increase in miR-126 expression
drives proliferation and activation of mast cells,40 functionally
linking our observations of increased miR-126 expression with
an increase in mast cell–related gene expression in the DKO
megakaryocytes. Reports from zebrafish categorize c-Myb as a
direct target of miR-126. Morpholino knockdown of miR-126
results in increased c-Myb expression, which promotes red cell
production at the expense of thrombocytes.41 Overexpression of
miR-126 in human embryonic stem-cell derived CD34⫹ cells
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 13 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 11
MKL2 IN MEGAKARYOCYTOPOIESIS
2327
Table 4. DKO and Srf Pf4-cKO megakaryocytes have differential megakaryocyte transmembrane gene expression
FPKM
Gene
1110008F13Rik
WT
SRF cKO
MKL1 KO
MKL2 cKO
DKO
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
Cd47
30.43
31.40
25.75
23.65
0.00
Cd63
555.94
402.13
499.11
485.78
921.27
Cd9
33.04*
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*
126.13
68.79
80.62
146.31
91.92
Esam
Eya2
31.29
41.15
24.88
10.51*
Fads2
207.86
130.48
14.01*
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
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
Itm2b
691.15
965.94
734.92
600.35
20.66*
50.74*
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
Mpl
92.44
57.96
116.22
Nrgn
437.18
256.78
303.27
P2rx1
135.55
63.86*
119.27
102.96
Pdzk1ip1
133.50
68.47
107.33
125.34
40.82*
203.67
57.66
23.74*
226.77
35.34*
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
Slc25a3
413.05
291.79
320.12
306.51
Slc35d3
84.72
40.63
53.39
101.96
Slc37a2
2.20
6.28
1.83
0.80
Slc6a4
28.19*
428.44
18.50*
1.74
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 ⬍ 0.05) compared to WT.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2328
BLOOD, 13 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 11
SMITH et al
Table 5. Top genes increased in both DKO and Srf Pf4-cKO
megakaryocytes
FPKM
Gene
WT
SRF cKO
MKL1 KO
MKL2 cKO
DKO
Camp
3.55
217.84
2.54
0.68
365.42
Cd177
0.62
25.74
0.49
0.29
19.06
Cebpe
0.99
38.18
1.03
0.48
78.54
Cldn15
2.83
50.78
3.05
1.93
51.11
Cma1
0.26
15.85
0.25
0.14
24.63
Cpa3
2.95
46.38
4.18
2.93
53.55
Ctsg
80.04
1110.46
77.54
42.09
1436.34
Dmkn
4.20
30.77
4.87
2.36
73.51
Elane
126.51
2657.87
129.82
76.19
4523.18
Epx
1.39
16.91
0.79
1.00
21.83
Fcnb
2.37
196.80
4.08
1.98
107.95
G0s2
0.41
10.25
0.43
0.20
15.04
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
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
Mgst2
5.72
104.82
3.61
2.69
158.53
Mogat2
0.48
33.16
0.44
0.46
19.44
Ms4a3
33.51
348.80
38.93
19.18
483.45
Ngp
9.93
764.69
5.76
2.56
834.41
Oscp1
0.74
10.77
0.75
0.74
13.66
Pglyrp1
0.86
79.38
1.25
0.59
204.23
Prg2
19.58
220.00
19.20
14.11
858.80
Prg3
2.13
17.73
1.91
1.44
71.50
Prss34
3.89
81.31
5.31
2.95
273.43
Prss57
Prtn3
0.95
36.18
1.00
0.60
76.53
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
Saa3
606.97
3825.61
628.93
244.01
9136.90
Slpi
30.75
244.71
36.52
17.42
663.24
Tst
1.56
15.88
1.14
1.26
35.82
A list of genes more than 16-fold increased in DKO and Srf Pf4-cKO megakaryocytes compared to WT megakaryocytes. 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.
leads to a decrease in erythroid colony formation; megakaryocyte potential was not assessed by this report.42 However,
expression of miR-126 decreases with megakaryocyte differentiation from adult CD34⫹ cells.43 Lastly, several groups have
reported the aberrant expression of miR-126 in acute myeloid
leukemia-initiating cells.43-45 These reports suggest that deregulation of miR-126 and other miRs (eg, miR-155) may be the
additional hits that drive leukemic progression. This suggests an
intriguing hypothesis in the case of t(1;22) AMKL; the fusion
protein OTT-MKL1 may disrupt the balance of TCFs and
MRTFs at the Egfl7 promoter similar to the DKO mice. The
result is the production of hyperproliferative cells that have
impaired megakaryocyte differentiation because of lack of
normal MKL1 expression. This is also supported by competitive
repopulation experiments in which overexpression of miR-126
provides an engraftment advantage.46 These findings reveal a
connection between the MRTFs, Ets domain proteins, and
miR-126 highlighting a pathway that may give insight into the
pathogenesis of AMKL.
Acknowledgments
The authors thank Dr Eric Olson for the Mkl2 floxed mice, Dr
Stephen Morris for the Mkl1 KO mice, and Stephanie Donaldson for excellent mouse husbandry. The authors also thank Dr
Emanuela Bruscia, Alexandra Teixeira, Dr Betty Lawton, and Dr
Dennis Jones for thoughtful insights and careful editing of this
manuscript.
This work was supported by National Institutes of Health (NIH)
grant F31 HL 094118 (to E.C.S.), and by NIH grants DK086267,
DK072442 (Yale Center of Excellence in Molecular Hematology), and
the Connecticut Stem Cell Fund (to D.S.K.), and HL106184 (to P.G.).
Authorship
Contribution: E.C.S. designed and performed experiments and
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.
performed bioinformatics analysis; S.H., P.G., and J.E.I. contributed scientific knowledge; and D.S.K. provided mentorship and
intellectual input, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Diane S. Krause, Department of Laboratory
Medicine, Yale University School of Medicine, PO Box 208073,
333 Cedar St, New Haven, CT 06520-8073; e-mail: diane.krause@
yale.edu.
References
1. Ma Z, Morris SW, Valentine V, et al. Fusion of two
novel genes, RBM15 and MKL1, in the t(1;22)
(p13;q13) of acute megakaryoblastic leukemia.
Nat Genet. 2001;28(3):220-221.
2. Mercher T, Busson-Le Coniat M, Nguyen Khac F,
et al. Recurrence of OTT-MAL fusion in t(1;22) of
infant AML-M7. Genes Chromosomes Cancer.
2002;33(1):22-28.
3. Ma X, Renda MJ, Wang L, et al. Rbm15 modulates Notch-induced transcriptional activation and
affects myeloid differentiation. Mol Cell Biol.
2007;27(8):3056-3064.
the c-fos serum response element. Cell. 1994;
76(2):411.
6. Arsenian S, Weinhold B, Oelgeschlager M,
Ruther U, Nordheim A. Serum response factor is
essential for mesoderm formation during mouse
embryogenesis. EMBO J. 1998;17(21):62896299.
7. Sun Y, Boyd K, Xu W, et al. Acute myeloid
leukemia-associated Mkl1 (Mrtf-a) is a key regulator of mammary gland function. Mol Cell Biol.
2006;26(15):5809-5826.
4. Wang DZ, Li S, Hockemeyer D, et al. Potentiation
of serum response factor activity by a family of
myocardin-related transcription factors. Proc Natl
Acad Sci U S A. 2002;99(23):14855-14860.
8. Oh J, Richardson JA, Olson EN. Requirement of
myocardin-related transcription factor-B for remodeling of branchial arch arteries and smooth
muscle differentiation. Proc Natl Acad Sci U S A.
2005;102(42):15122-15127.
5. Dalton S, Treisman R. Characterization of SAP-1,
a protein recruited by serum response factor to
9. Li S, Wang DZ, Wang Z, Richardson JA, Olson EN.
The serum response factor coactivator myocardin
is required for vascular smooth muscle development. Proc Natl Acad Sci U S A. 2003;100(16):
9366-9370.
10. Cen B, Selvaraj A, Burgess RC, et al. Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required
for serum induction of SRF target genes. Mol Cell
Biol. 2003;23(18):6597-6608.
11. Du KL, Chen M, Li J, Lepore JJ, Mericko P,
Parmacek MS. Megakaryoblastic leukemia
factor-1 transduces cytoskeletal signals and induces smooth muscle cell differentiation from undifferentiated embryonic stem cells. J Biol Chem.
2004;279(17):17578-17586.
12. Tabuchi A, Estevez M, Henderson JA, et al.
Nuclear translocation of the SRF co-activator
MAL in cortical neurons: role of RhoA signalling.
J Neurochem. 2005;94(1):169-180.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 13 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 11
13. Cheng EC, Luo Q, Bruscia EM, et al. Role for
MKL1 in megakaryocytic maturation. Blood.
2009;113(12):2826-2834.
14. Halene S, Gao Y, Hahn K, et al. Serum response
factor is an essential transcription factor in megakaryocytic maturation. Blood. 2010;116(11):19421950.
15. Ragu C, Boukour S, Elain G, et al. The serum
response factor (SRF)/megakaryocytic acute leukemia (MAL) network participates in megakaryocyte development. Leukemia. 2010;24(6):12271230.
reveals novel transmembrane proteins in human
platelets and mouse megakaryocytes including
G6b-B, a novel immunoreceptor tyrosine-based
inhibitory motif protein. Mol Cell Proteomics.
2007;6(3):548-564.
25. Chintala S, Tan J, Gautam R, et al. The Slc35d3
gene, encoding an orphan nucleotide sugar
transporter, regulates platelet-dense granules.
Blood. 2007;109(4):1533-1540.
26. Meng R, Wang Y, Yao Y, et al. SLC35D3 delivery
from megakaryocyte early endosomes is required
for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models. Blood. 2012;120(2):404-414.
16. Mokalled MH, Johnson A, Kim Y, Oh J, Olson EN.
Myocardin-related transcription factors regulate
the Cdk5/Pctaire1 kinase cascade to control neurite outgrowth, neuronal migration and brain development. Development. 2010;137(14):23652374.
27. Nichol D, Shawber C, Fitch MJ, et al. Impaired
angiogenesis and altered Notch signaling in mice
overexpressing endothelial Egfl7. Blood. 2010;
116(26):6133-6143.
17. Tiedt R, Schomber T, Hao-Shen H, Skoda RC.
Pf4-Cre transgenic mice allow the generation of
lineage-restricted gene knockouts for studying
megakaryocyte and platelet function in vivo.
Blood. 2007;109(4):1503-1506.
28. Subramanian A, Tamayo P, Mootha VK, et al.
Gene set enrichment analysis: a knowledgebased approach for interpreting genome-wide
expression profiles. Proc Natl Acad Sci U S A.
2005;102(43):15545-15550.
18. Pronk CJ, Rossi DJ, Mansson R, et al. Elucidation of the phenotypic, functional, and molecular
topography of a myeloerythroid progenitor cell
hierarchy. Cell Stem Cell. 2007;1(4):428-442.
29. Mootha VK, Lindgren CM, Eriksson KF, et al.
PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34(3):
267-273.
19. Schwer HD, Lecine P, Tiwari S, Italiano JE Jr,
Hartwig JH, Shivdasani RA. A lineage-restricted
and divergent beta-tubulin isoform is essential for
the biogenesis, structure and function of blood
platelets. Curr Biol. 2001;11(8):579-586.
20. Lecine P, Italiano JE Jr, Kim SW, Villeval JL,
Shivdasani RA. Hematopoietic-specific beta 1
tubulin participates in a pathway of platelet biogenesis dependent on the transcription factor NFE2. Blood. 2000;96(4):1366-1373.
21. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25(9):1105-1111.
22. Trapnell C, Williams BA, Pertea G, et al. Transcript assembly and quantification by RNA-Seq
reveals unannotated transcripts and isoform
switching during cell differentiation. Nat Biotechnol. 2010;28(5):511-515.
23. Chen C, Fuhrken PG, Huang LT, et al. A systemsbiology analysis of isogenic megakaryocytic and
granulocytic cultures identifies new molecular
components of megakaryocytic apoptosis. BMC
Genomics. 2007;8:384.
24. Senis YA, Tomlinson MG, Garcia A, et al. A comprehensive proteomics and genomics analysis
30. Wang S, Aurora AB, Johnson BA, et al. The
endothelial-specific microRNA miR-126 governs
vascular integrity and angiogenesis. Dev Cell.
2008;15(2):261-271.
31. Wei K, Che N, Chen F. Myocardin-related transcription factor B is required for normal mouse
vascular development and smooth muscle gene
expression. Dev Dyn. 2007;236(2):416-425.
32. Gilles L, Bluteau D, Boukour S, et al. MAL/SRF
complex is involved in platelet formation and
megakaryocyte migration by regulating MYL9
(MLC2) and MMP9. Blood. 2009;114(19):42214232.
33. Gao Y, Smith E, Ker E, et al. Role of RhoAspecific guanine exchange factors in regulation of
endomitosis in megakaryocytes. Dev Cell. 2012;
22(3):573-584.
34. Cooper SJ, Trinklein ND, Nguyen L, Myers RM.
Serum response factor binding sites differ in three
human cell types. Genome Res. 2007;17(2):136144.
35. Tatsumoto T, Xie X, Blumenthal R, Okamoto I,
Miki T. Human ECT2 is an exchange factor for
Rho GTPases, phosphorylated in G2/M phases,
MKL2 IN MEGAKARYOCYTOPOIESIS
2329
and involved in cytokinesis. J Cell Biol. 1999;
147(5):921-928.
36. Jurak Begonja A, Hoffmeister KM, Hartwig JH,
Falet H. FlnA-null megakaryocytes prematurely
release large and fragile platelets that circulate
poorly. Blood. 2011;118(8):2285-2295.
37. Asparuhova MB, Ferralli J, Chiquet M,
Chiquet-Ehrismann R. The transcriptional regulator megakaryoblastic leukemia-1 mediates serum
response factor-independent activation of
tenascin-C transcription by mechanical stress.
FASEB J. 2011;25(10):3477-3488.
38. Harris TA, Yamakuchi M, Kondo M, Oettgen P,
Lowenstein CJ. Ets-1 and Ets-2 regulate the expression of microRNA-126 in endothelial cells.
Arterioscler Thromb Vasc Biol. 2010;30(10):19901997.
39. Wang Z, Wang DZ, Hockemeyer D, McAnally J,
Nordheim A, Olson EN. Myocardin and ternary
complex factors compete for SRF to control
smooth muscle gene expression. Nature. 2004;
428(6979):185-189.
40. Ishizaki T, Tamiya T, Taniguchi K, et al. miR126
positively regulates mast cell proliferation and
cytokine production through suppressing Spred1.
Genes Cells. 2011;16(7):803-814.
41. Grabher C, Payne EM, Johnston AB, et al. Zebrafish microRNA-126 determines hematopoietic
cell fate through c-Myb. Leukemia. 2011;25(3):
506-514.
42. Huang X, Gschweng E, Van Handel B, Cheng D,
Mikkola HK, Witte ON. Regulated expression of
microRNAs-126/126* inhibits erythropoiesis from
human embryonic stem cells. Blood. 2011;117(7):
2157-2165.
43. Garzon R, Pichiorri F, Palumbo T, et al. MicroRNA
fingerprints during human megakaryocytopoiesis.
Proc Natl Acad Sci U S A. 2006;103(13):50785083.
44. Cammarata G, Augugliaro L, Salemi D, et al. Differential expression of specific microRNA and
their targets in acute myeloid leukemia. Am J
Hematol. 2010;85(5):331-339.
45. Li Z, Lu J, Sun M, et al. Distinct microRNA expression profiles in acute myeloid leukemia with
common translocations. Proc Natl Acad Sci
U S A. 2008;105(40):15535-15540.
46. O’Connell RM, Chaudhuri AA, Rao DS,
Gibson WS, Balazs AB, Baltimore D. MicroRNAs
enriched in hematopoietic stem cells differentially
regulate long-term hematopoietic output. Proc
Natl Acad Sci U S A. 2010;107(32):14235-14240.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
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 (3446 articles)
Platelets and Thrombopoiesis (744 articles)
Thrombocytopenia (234 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.