Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer that controls the differentiation and function of various cellular systems, including hematopoietic cells. miR-142 is one of the most prevalently expressed miRNAs within the hematopoietic lineage. To address the in vivo functions of miR-142, we utilized a novel reporter and a loss-of-function mouse allele that we have recently generated. In this study, we show that miR-142 is broadly expressed in the adult hematopoietic system. Our data further reveal that miR-142 is critical for megakaryopoiesis. Genetic ablation of miR-142 caused impaired megakaryocyte maturation, inhibition of polyploidization, abnormal proplatelet formation, and thrombocytopenia. Finally, we characterized a network of miR-142-3p targets which collectively control actin filament homeostasis, thereby ensuring proper execution of actin-dependent proplatelet formation. Our study reveals a pivotal role for miR-142 activity in megakaryocyte maturation and function, and demonstrates a critical contribution of a single miRNA in orchestrating cytoskeletal dynamics and normal hemostasis.https://doi.org/10.7554/eLife.01964.001
DNA carries all the information needed for life. This includes the codes required for making proteins, as well as instructions on when, where, and how much of these proteins need to be produced. There are a number of ways by which cells control protein manufacturing, one of which is based on small RNAs called microRNAs. Before proteins are assembled, the DNA molecule is copied into a temporary replica dubbed messenger RNA. microRNAs are able to recognize specific messenger RNA molecules and block protein production.
microRNAs serve a very important regulatory role in our bodies and are involved in virtually all cellular processes, including the production of all classes of blood and immune cells. Platelets seal injuries and prevent excessive bleeding by creating a clot at the location of a wound. Platelets are produced in huge cellular factories called megakaryocytes, which need to have a flexible and dynamic internal skeleton or cytoskeleton to produce the platelets.
Chapnik et al. focus on one specific microRNA gene, which is vital for the production and the function of several classes of blood and immune cells. Chapnik et al. created a mouse model that does not produce one specific microRNA—miR-142—and found that mutant mice produced fewer platelets than normal mice. Although one possible explanation for this is that the mutant mice also had fewer megakaryocytes than normal, Chapnik et al. unexpectedly found that the number of megakaryocytes was in fact higher. However, these megakaryocytes do not reach functional maturity, which is required for platelet production. Many of the megakaryocytes made by the mutant mice were also smaller than normal and had an unusual cytoskeleton.
Using a genomic approach and molecular tools, Chapnik et al. show that miR-142 affects the production of several of the proteins responsible for the dynamic flexibility of the cytoskeleton in mature megakaryocytes. Therefore, a single microRNA can target multiple different proteins that coordinate the same pathway in the cells that are critical for clotting and hence for human health.
miR-142 has also been suggested to have important functions in blood stem cells and in blood cancer (leukemia). Therefore, the new mouse model could be used to investigate many other facets of the blood and immune system. Further research could also focus on whether the same cytoskeletal network is in charge of miR-142 activity in other blood cells, or if miR-142 silences different targets in different cells.https://doi.org/10.7554/eLife.01964.002
microRNAs (miRNAs) are single-stranded RNA molecules of 22 nucleotides in length, processed from endogenous hairpin transcripts. miRNAs provide cells with a sequence-based silencing mechanism through base-pairing of a minimal recognition sequence, called the miRNA ‘seed’ (Bartel, 2009; Carthew and Sontheimer, 2009; Fabian et al., 2010).
miRNAs control hematopoiesis and the function of both lymphoid and myeloid progeny (Chen and Lodish, 2005; Lawrie, 2007; Garzon and Croce, 2008; Navarro and Lieberman, 2010). For example, in vivo studies uncovered roles for miR-451 in erythropoiesis (Patrick et al., 2010; Rasmussen et al., 2010; Yu et al., 2010), for miR-223 in granulopoiesis (Johnnidis et al., 2008), for miR-150 in the commitment of multipotent myeloerythroid progenitors (Lu et al., 2008), and for miR-155 in the mammalian immune system (Rodriguez et al., 2007; Thai et al., 2007; Mann et al., 2010; O'Connell et al., 2010, 2007). Megakaryocytes (MKs) display a distinctive miRNA expression pattern (Garzon et al., 2006; Opalinska et al., 2010; Xu et al., 2012). However, functional genetic studies dissecting the role of miRNA in megakaryopoiesis are still limited.
In the present work, we focused on miR-142, a hematopoietic-specific miRNA, which resides in a genomic locus that was previously associated with t(8;17) translocation in B-cell leukemia (Gauwerky et al., 1989). Pioneering experimental evidence has suggested miR-142 involvement in lymphocyte differentiation (Chen et al., 2004) and recently, miR-142 was also shown to play a role in the specification of definitive hemangioblasts (Lu et al., 2013; Nimmo et al., 2013), and in lymphoid and myeloid lineages (Gauwerky et al., 1989; Chen et al., 2004; Huang et al., 2009; Belz, 2013; Lagrange et al., 2013; Lu et al., 2013; Nimmo et al., 2013; Sonda et al., 2013; Sun et al., 2013; Zhou et al., 2013). Furthermore, miR-142 is involved in the compound immune response to North American eastern equine encephalitis virus (Trobaugh et al., 2014) and our work uncovered a key role for miR-142 in the maintenance of CD4+ dendritic cells (Mildner et al., 2013).
MK maturation is an intricate process that involves DNA replication in the absence of cytoplasmic division, termed endomitosis. Polyploid MKs also exhibit distinctive cytoskeletal rearrangements that enable the biosynthesis of platelets (also known as thrombocytes). The elaborated actin cytoskeleton of MKs is uniquely organized in a way that allows cytoplasmic proplatelet protrusions to bend and bifurcate into multiple tips, from which platelets are subsequently released to the bloodstream. Dysregulation of the actin cytoskeleton impairs the generation of mature platelets (Hartwig and Italiano, 2006; Bender et al., 2010). Accordingly, actin-modulating genes were shown to be crucial for MK maturation and have been implicated in the etiology of platelet-related disorders (Villa et al., 1995; Kajiwara et al., 1999).
We characterize miR-142-deficient mice that display an array of hematological defects, including pronounced thrombocytopenia. We show that miR-142 controls multiple facets of MK differentiation including control of cell size, ploidy, and proplatelet elaboration. Furthermore, we demonstrate that miR-142-3p controls platelet biosynthesis by orchestrating the coordinated expression of several distinct nodes in a network of actin cytoskeleton regulators. Our study reveals a novel miRNA-dependent circuit that maintains cytoskeletal integrity, and suggests that a single miRNA may broadly regulate cell function by controlling a coherent set of effectors in a given pathway.
A miR-142−/− allele was created by insertion of an exogenous gene trap sequence ∼50 bp upstream of the murine pre-miR-142. This cassette disrupted normal transcription and drove the expression of a beta-galactosidase reporter gene (Stanford et al., 2001; Hansen et al., 2008; Osokine et al., 2008; Figure 1A).
To confirm miR-142 nullification, we collected circulating mononuclear cells from peripheral blood of homozygous mutant mice and wild-type (WT) littermates. miR-142-3p is the guide strand from the miR-142 hairpin, whereas the sister ‘passenger’ miR-142-5p strand is expressed in negligible levels (Nimmo et al., 2013; Figure 1B). The expression of both miR-142-3p and miR-142-5p was abolished in miR-142−/− circulating blood cells as exemplified by quantitative real-time PCR (qPCR, Figure 1B).
Genotyping the progeny of miR-142+/− intercrosses at embryonic day 14.5 (E14.5) revealed the predicted Mendelian distribution of miR-142+/+, miR-142+/−, and miR-142−/− embryos. However, postnatal survival at 3 weeks of age of miR-142 homozygous offsprings was lower than expected (18% instead of 25%), demonstrating that roughly a third of miR-142−/− mice died perinatally (Figure 1—source data 1). Surviving miR-142−/− mice did not display overt physical abnormalities, were fertile, and bred normally.
To comprehensively characterize miR-142 expression pattern, we performed a fluorescence-based semi-quantitative detection of beta-galactosidase activity in viable hematopoietic cells. In accordance with previous reports (Lagos-Quintana et al., 2002; Chen et al., 2004; Ramkissoon et al., 2006; Merkerova et al., 2008), this study revealed pan-hematopoietic activity of the miR-142 promoter, which drove the expression of a LacZ transgene from the endogenous miR-142 locus in all lymphoid and myeloid lineages examined (Figure 1C).
To assess the impact of miR-142 loss in vivo, we performed complete blood counts that revealed reduced numbers of erythrocytes and white blood cells in miR-142−/− animals relative to WT littermates (Figure 1D,E). Interestingly, at 2 months of age, miR-142−/− mice displayed a striking ∼50% decrease in platelet counts and a ∼10% increase in mean platelet volume (MPV), relative to controls (Figure 1F,G). Thus, miR-142−/− animals suffer from macrothrombocytopenia. The reduction in platelet numbers and the concordant increase in platelet size were even more pronounced when analyzed at 1 year of age (Figure 1F,G).
In addition, lethally irradiated WT mice, which were reconstituted with miR-142−/− bone marrow (BM) cells and analyzed 6 weeks following transfer, showed platelet paucity relative to controls that were reconstituted with WT BM cells (Figure 1—figure supplement 1). However, the decrease in platelet numbers in this model was less significant compared to that demonstrated in germline miR-142-deficient mice (Figure 1F), plausibly due to contribution of WT cells. Taken together, these data demonstrate that hematopoietic-specific miR-142 activity is required for normal platelet production.
We hypothesized that the diminished numbers of circulating platelets might stem from a defect in the development of MKs. First, we confirmed that miR-142-3p is the functional dominant ‘guide’ strand in MKs, and that both sister miR-142 species are nullified in miR-142−/− MKs (Figure 2—figure supplement 1A). We also confirmed that the expression of Bzrap1, a regulator of synaptic transmission (Chardenot et al., 2002; Mittelstaedt and Schoch, 2007) positioned 3.5 Kb downstream of miR-142, is unchanged in miR-142−/− MKs (Figure 2—figure supplement 1B).
To gain insight into the impact of miR-142 nullification on early MK development, we performed a high-resolution flow cytometry assay for the characterization of myeloerythroid progenitors (Pronk et al., 2007; Pronk and Bryder, 2011). The numbers of miR-142−/− bipotent MK-erythroid precursors (PreMegEs) were marginally increased relative to controls (Figure 2A,B). In contrast, the direct descendants of PreMegEs, namely the unipotent MK-progenitors (MkPs), which further give rise to mature MKs, were significantly increased in miR-142−/− animals, relative to WT controls (Figure 2A,B). Intriguingly, the expression levels of regulatory markers of MK development remained largely unchanged in miR-142−/− PreMegEs and MkPs, relative to WT controls (Figure 2C,D).
The observed expansion of MkPs in miR-142 mutants was further supported by a concordant elevation in the numbers of CD41+/CD42+ miR-142−/− MKs relative to WT controls (Figure 2E).
We next investigated the numbers of MKs in situ, by immunohistochemical staining of femoral BM sections against von Willebrand Factor (vWF), which is specifically expressed in MKs from an early stage of differentiation (Tomer, 2004). miR-142−/− mice displayed a ∼50% increase in the numbers of vWF-positive MKs, relative to WT littermates (Figure 2F). Noteworthy, miR-142−/− mice exhibited splenomegaly and a marked elevation in the number of splenic MKs, relative to control littermates, suggesting extramedullary thrombopoiesis (Figure 2—figure supplement 2).
To further confirm the expansion of miR-142−/− MkPs, we employed a colony forming unit-megakaryocyte (CFU-MK) assay that quantifies the MkP numbers in the BM. We observed higher numbers of CFU-MK colonies of miR-142−/− BMs, relative to WT controls (Figure 2G, right panel). Furthermore, each miR-142−/− colony typically harbored more cells than control colonies (Figure 2G, left panel).
To elucidate whether MkP expansion represented a cell-intrinsic phenomenon, we employed a competitive repopulation experiment (Figure 2H). Thus, we injected CD45.2+/miR-142−/− and congenic CD45.1+/WT BM cells in equal numbers into lethally irradiated CD45.1+ recipient mice. The reconstituted BM populations were analyzed for 6 weeks following transplantation. Cells expressing the pan-B-cell marker, B220, were equally represented by CD45.2+/miR-142−/− and CD45.1+/WT genotypes and served as engraftment controls. CD45.2+/miR-142−/− PreMegE levels showed a mild increase relative to CD45.1+/WT counterparts, confirming that this population is not appreciably affected by the loss of miR-142 (Figure 2I,J). In contrast, CD45.2+/miR-142−/− MkPs were over-represented in chimeric BMs at a ratio of ∼5:1 relative to CD45.1+/WT MkPs (Figure 2I,J). Thus, the MkP expansion observed in miR-142−/− BM is cell-autonomous. Taken together, these data suggest that miR-142 activity regulates the differentiation of the MK lineage in a cell-intrinsic manner.
The observed elevation in miR-142−/− MK frequency was unexpected, because increased MK numbers are usually correlated with higher platelet counts (Schafer, 2004). Thus, since the pronounced thrombocytopenia in miR-142-deficient mice was not caused due to a lack of MKs, we hypothesized that it may result from a block in MK maturation. An initial clue that miR-142−/− MKs were premature, came from the observation that the average size of vWF-positive miR-142−/− MKs in the femoral BM was smaller than that of WT MKs. Indeed, miR-142−/− MKs showed a ∼25% reduction in sectional area, relative to WT counterparts (Figure 3A,B).
We then differentiated MKs from E14.5 fetal liver (FL), under defined ex vivo conditions, as previously described (Shivdasani and Schulze, 2005). After 4 days in culture, FL-derived miR-142−/− MKs exhibited smaller cell size, compared to control MKs (Figure 3C,D).
Polyploidization is an additional important feature of MK maturity, which is associated with effective platelet production (Levine et al., 1982; Mattia et al., 2002; Ravid et al., 2002; Lee et al., 2009). We therefore tested the number of MK nuclei by flow cytometry. CD41+ BM-derived miR-142−/− MKs, exhibited reduced overall ploidy (Figure 3E). Furthermore, the fraction of mature (≥16N) miR-142−/− MKs was significantly diminished, relative to WT controls, whereas the percentage of low ploidy immature MK forms (≤8N) was higher in miR-142−/− BM (Figure 3E). Similar data were gained by ex vivo differentiation of FL-derived MKs, whereby high ploidy number (>32) was observed in only 4% of the miR-142−/− MK, relative to 12% in control MKs (Figure 3F). Thus, miR-142 is essential for normal endomitosis and reduced miR-142−/− platelet numbers might result from accumulation of immature, low-polyploid MKs that are poor producers of platelets (Mattia et al., 2002).
Proplatelet formation (PPF) represents the final phase of MK maturation, culminating in platelet release into the bloodstream (Machlus and Italiano, 2013). To analyze whether miR-142 is involved in this process, we performed an ex vivo PPF study on FL-derived MKs. Remarkably, we observed a striking threefold reduction in miR-142−/− MKs that were extending proplatelets, relative to control MKs (Figure 3G). We next re-introduced miR-142-3p into differentiated MKs, using dsRNA mimetics (Figure 3—figure supplement 1). The introduction of miR-142-3p was sufficient to recapitulate WT PPF levels in cells that are genetically miR-142-deficient (Figure 3H). Conversely, overexpression of miR-142-3p mimics in WT MKs did not yield any significant increase in PPF levels. Thus, miR-142-3p activity is essential for proper MK maturation and its loss results in defective platelet biogenesis. Furthermore, since re-introduction of miR-142-3p into miR-142−/− differentiated MKs restored functional identity, we conclude that there might be a continuous requirement for miR-142-3p activity to maintain MK maturity.
Dynamic rearrangement and organization of MK cytoskeletal structures is essential for normal megakaryopoiesis, endomitosis, and platelet production (Hartwig and Italiano, 2006; Lordier et al., 2008; Thon and Italiano, 2012). We therefore sought to characterize by confocal microscopy filamentous actin (F-actin) and tubulin in FL-derived MKs that were allowed to adhere to fibronectin for 3 hr. This analysis revealed a markedly disturbed cytoskeletal organization in miR-142−/− MKs compared to WT counterparts (Figure 4A) and mutant cells displayed a more immature circular profile than WT counterparts (Figure 4B).
Accordingly, ImagestreamX flow cytometery revealed a lower ratio of filamentous to globular (F/G) actin in miR-142−/− CD61+ MKs than in control MKs (Figure 4C,D). This analysis reaffirmed that miR-142−/− MKs are smaller in size, more circular and exhibited a more homogenous F-actin distribution than controls (Figure 4E–G).
Finally, we performed a cytochalasin D (CytoD) washout study, which followed the re-assembly of actin filaments after forced depolymerization (Figure 4H). The number of miR-142−/− MKs that were able to create stress-fibers following treatment with CytoD was significantly lower, relative to control MKs (Figure 4I). Collectively, these data reveal that miR-142 is pivotal for normal actin dynamics and architecture in MKs.
To determine the molecular mechanism for miR-142-mediated control of megakaryocytic development, we performed a genome-wide study for the identification of differentially expressed genes in miR-142−/− vs WT differentiated MKs. This study revealed that roughly 800 mRNAs were significantly deregulated due to loss of miR-142 (Figure 5A). Sylamer analysis (van Dongen et al., 2008) of microarray data, from WT and miR-142−/− FL-derived MKs, uncovered a highly significant enrichment for the 7- and 8-mer seeds of miR-142-3p among genes that were up-regulated in miR-142−/− MKs (Blue lines; Figure 5B). However, such enrichment was not evident for any other miRNA including the sister miRNA, miR-142-5p (Green lines; Figure 5B), further substantiating that miR-142-3p is the dominant functional miRNA from the pre-miR-142 hairpin. Accordingly, we depicted significant up-regulation of miR-142-3p targets within the set of TargetScan predicted targets (Friedman et al., 2009; Figure 5—figure supplement 1).
Gene ontology (GO) analysis using the database for annotation, visualization, and integrated discovery (DAVID) algorithm (Huang da et al., 2009a, 2009b) revealed perturbations of a variety of cellular processes in miR-142−/− MKs (Figure 5—source data 1). To enhance the power of our analysis, we next focused on targets that were commonly up-regulated in miR-142−/− MKs as well as in miR-142−/− CD24+ and CD24- in vitro-derived DCs that we previously characterized (Mildner et al., 2013). Forty genes were found to be commonly up-regulated in all three miR-142−/− hematopoietic cell types (Figure 5C, Figure 5—source data 2). This list is highly enriched with direct miR-142-3p putative targets (12 out of 40 genes). Subsequent DAVID analysis performed on these genes uncovered a significant enrichment for actin- and cytoskeleton-related functional GO categories (Figure 5C). Indeed, half of the dozen predicted miR-142-3p targets encode for pivotal actin cytoskeleton-associated proteins, including Cofilin-2 (Cfl2), Glucocorticoid receptor DNA binding factor 1 (Grlf1), Biorientation of chromosomes in cell division 1 (Bod1), Integrin alpha V (Itgav), Twinfilin-1 (Twf1), and Wiskott–Aldrich syndrome-like (Wasl). Intriguingly, the latter was recently suggested as a potential target of miR-142-3p in the process of actin-mediated mycobacterial infection (Bettencourt et al., 2013). The up-regulation of these six miR-142-3p targets in miR-142−/− MKs, was further confirmed by qPCR (Figure 5D). Among these targets, Cfl2 expression was also found to be up-regulated in miR-142−/− MkPs (Figure 5—figure supplement 2). Introducing of miR-142-3p into differentiated MKs, using dsRNA mimetics, was sufficient to normalize the expression levels of representative miR-142-3p targets, Wasl and Cfl2 in miR-142−/− MKs (Figure 5E). Western blot analysis revealed up-regulation of the protein products of Wasl and Cfl2 in miR-142−/− MKs, further substantiating them as bona fide miR-142-3p targets (Figure 5F–G). Using luciferase reporter assays, we showed that the mRNAs of Wasl, Cfl2, Grlf1, Itgav and Twf1 are directly targeted by miR-142-3p (Figure 5H–J, Figure 5—figure supplements 3 and 4). Furthermore, the quantitative contributions of individual binding sites within the target 3′ UTR was revealed by stepwise loss of miR-142 regulation, when miR-142-3p binding sites were sequentially mutated (Figure 5H–J, Figure 5—figure supplements 3 and 4). Taken together, we discovered that the expression of a compound set of actin cytoskeleton regulators is post-transcriptionally controlled by miR-142-3p.
miR-142-deficient MKs displayed perturbed actin filament dynamics and diminished proplatelet formation. This is presumably due to de-repression of several actin cytoskeleton components, including Wasl, Cfl2, Twf1, Itgav or Grlf1, which are all direct miR-142-3p targets. Thus, we hypothesized that knocking down these miR-142-3p targets may relieve the PPF defect in miR-142−/− MKs. For this purpose, we transduced FL-derived MKs with short hairpin RNA (shRNA)-expressing lentiviral vectors that effectively knocked-down targets with more than three miR-142-3p binding sites, namely Wasl, Cfl2 or Grlf1 (shWasl, shCfl2 and shGrlf1, respectively, Figure 6—figure supplement 1). A lentivirus encoding a shRNA directed against RFP (shRFP) was employed as a control. Following transduction, differentiation of MKs was induced and PPF was quantified 3 days later. The PPF defect was clearly evident in miR-142−/− MKs transduced with shRFP (Figure 6A,B). In contrast, miR-142−/− MKs that were transduced with shRNA directed against Cfl2, Wasl, Grlf1, displayed a prominent elevation of PPF levels, and approximated levels observed in control WT MKs transduced with shRFP (Figure 6A,B). This strong elevation in PPF levels was limited to miR-142−/− MKs, whereas in WT MKs, knockdown of miR-142-3p targets did not significantly alter PPF levels, except for Wasl-targeting shRNA that exhibited a modest increase in PPF efficacy following transduction (Figure 6—figure supplement 2A). Additional pairwise target comparison revealed compound relationships between miR-142-3p targets (Figure 6C; Figure 6—figure supplement 2B). Remarkably, knockdown of both Cfl2 and Wasl, which carry out opposing functions within the actin regulatory network, did not yield any significant increase in PPF levels in miR-142-deficient MKs (Figure 6C). Conversely, concomitant Cfl2 and Grlf1 knockdown, two proteins that are important for destabilizing actin polymers, enhanced PPF in miR-142 null MKs (Figure 6C). Lastly, a pool of shRNA against miR-142-3p targets was able to fully restore PPF capacity to WT levels (Figure 6A–C). These data demonstrate that the expression of a set of actin cytoskeleton regulators should be tightly orchestrated by miR-142-3p in order to effectively promote platelet biogenesis.
Hematopoietic-specific miR-142 has emerged in recent years as a critical regulator of various blood and lymphoid cell lineages (Gauwerky et al., 1989; Chen et al., 2004; Huang et al., 2009; Belz, 2013; Lagrange et al., 2013; Lu et al., 2013; Mildner et al., 2013; Nimmo et al., 2013; Sonda et al., 2013; Sun et al., 2013; Zhou et al., 2013). Our analysis unveils the critical functions of miR-142 in the MK lineage. Using a recently-established mouse model, we show that deletion of the miR-142 allele results in pronounced thrombocytopenia. Our in vivo studies and culture assays reveal that proper miR-142 function is essential, in a cell-intrinsic manner, for MK maturation, including control of cell size, polyploidization and proplatelet elaboration.
Furthermore, MKs require sustained miR-142-3p expression, as re-introduction of synthetic miR-142-3p mimetics, even onto differentiated MKs, was sufficient to restore functional maturity.
Mechanistically, we demonstrate that miR-142-3p mediates the repression of an interconnected set of actin cytoskeleton regulators. These collectively contribute to MK maturation and their dysregulation is responsible for incomplete maturation observed in miR-142-deficient MKs. Very often, the sets of target genes that are predicted to be regulated by a particular miRNAs do not coalesce into coherent networks with distinct biological functions (Stark et al., 2005). However, our work provides a clear example of an individual miRNA that co-regulates a network of functionally-associated targets. Indeed, the ability of a miRNA to modulate the expression of multiple targets within the same pathway simultaneously was previously suggested in other cellular contexts. For instance, the bicistronic miR-143/145 miRNA gene cluster dictates smooth-muscle cell phenotypic switching by orchestrating the expression of a cadre of cytoskeletal remodeling regulators (Xin et al., 2009). Furthermore, miR-125 family members control hematopoietic stem cell pool size by targeting a cohort of proapoptotic genes (Guo et al., 2010; Ooi et al., 2010). Therefore, a single miRNA that cumulatively targets several nodes within the same biological circuit may serve as an effective means to control cellular behavior.
The actin cytoskeleton participates in a wide array of cellular functions, and the dynamic turnover between its F-actin and monomeric G-actin forms is regulated by a large number of actin-binding proteins. Within this cytoskeletal regulatory network, the set of genes targeted by miR-142-3p contains components with divergent functions. For example, Cfl2 and Twf1 participate in disassembly of actin filaments (Bamburg, 1999; Moseley et al., 2006). Likewise, Grlf1 is a GTPase activating protein that has been implicated in disruption of the organized actin cytoskeleton (McGlade et al., 1993). Wasl, on the other hand, promotes actin polymerization by catalyzing filament branching together with the Arp2/3 complex (Rotty et al., 2013). Thus, miR-142 deficiency destabilizes feedback loops required for actin filament homeostasis, stress fiber formation, and actin remodeling. This in turn impairs proplatelet generation and plausibly other MK-intrinsic cellular functions, such as endomitosis. It is also likely that additional miR-142-3p targets participate in the regulation of MK differentiation. For example, the over-representation of miR-142-deficient MkPs might result from a dysregulation of a distinct set of targets that are not necessarily related to actin regulation.
Repression of miR-142-3p actin-associated targets was found to be sufficient for restoring PPF levels in miR-142-deficient MKs. Interestingly, knocking down the same targets in WT MKs did not result in significant increase in PPF capacity over WT baseline levels. This might be related to a certain threshold in PPF levels that cannot be crossed, even when regulation of miR-142-3p is mimicked.
In summary, our analysis suggests a cardinal role for miR-142 in maturation of MKs, and in particular in controlling a network of chief actin regulators to facilitate MK terminal differentiation. The data challenges the prevailing paradigm, that miRNAs exert only subtle effects, often elicited by specific stressors (Ebert and Sharp, 2012; Mendell and Olson, 2012; Pelaez and Carthew, 2012), by providing in vivo evidence that genetic manipulation of a single miRNA may have a significant impact on cellular commitment and differentiation.
Several platelet disorders have been associated with mutations in genes involved in actin organization, including Wiskott-Aldrich syndrome protein (WASP) (Massaad et al., 2013) and Actinin alpha 1 (ACTN1) (Kunishima et al., 2013). Because miR-142 locus is involved in B-cell leukemogenesis (Gauwerky et al., 1989) and since miR-142 is necessary for CD4+ DCs development (Mildner et al., 2013) and MK differentiation, miR-142 may function as a broad hematopoietic pro-differentiation factor. Thus, changes in miR-142 levels or activity may lead to platelet disorders or to hematopoietic malignancies.
Mice strains were housed and handled in accordance with protocols approved by the Institutional Animal Care and Use Committee of WIS. To generate miR-142−/− mice, a gene-trapped embryonic stem cell clone (ES, C57BL/6J strain) from TIGM (College Station, TX), was chosen based on an insertion upstream of miR-142 hairpin. ES cells were microinjected into C57BL/6J host blastocysts. Chimeras and further transmission of the targeted allele through the male germline to heterozygous pedigree was confirmed by PCR analysis of genomic tail DNA. Homozygous and WT littermate mice were generated by additional intercrosses. For transplantation experiments, recipient mice were lethally irradiated, using a 10.5Gy cesium source. After 10 days of ciprofloxacin prophylaxis, approximately 5 × 106 cells were injected into the tail vein for repopulation of the hematopoietic system.
About 100 μl whole blood was retro-orbitally drawn from age- and sex-matched miR-142−/− and WT littermates into glass capillary tubes that were pre-treated with 5 μl of 0.5M EDTA, to prevent coagulation. Complete blood count was performed on ADVIA 120 Hematology System (Siemens Healthcare, Erlangen, Germany) by American Medical Laboratories (Herzliya, Israel).
Femora and spleens of age- and sex-matched miR-142−/− and WT littermates were excised after euthanasia and fixed overnight in 4% paraformaldehyde. Femora were then decalcified in 14% EDTA for 2–5 days. Specimens were dehydrated in graded ethanols, washed and processed into paraffin blocks. Longitudinal paraffin sections were stained with hematoxylin and eosin (H&E), May–Grünwald Giemsa or immunostained with anti-von Willebrand Factor (Dako, Agilent technologies, Santa Clara, CA). An Axioplan light microscope (Carl Zeiss, Oberkochen, Germany), equipped with an eyepiece graticule (grid), was utilized for quantification of MK numbers, per 20X magnification field, in 5 μm BM or spleen sections. MKs size was measured as the maximal diameter in 50 consecutive MKs in the BM of the distal femur, using the cellSens digital imaging software on an Olympus BX51 microscope at 40X magnification.
Mouse BM cells were harvested via flushing of the long bones with Dulbecco modified Eagle medium (DMEM; Invitrogen, Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Life Technologies, Carlsbad, CA), was followed by filtering through a 70-µm nylon mesh cell strainer, to remove bone debris. BM mononuclear cells were cultured in MethoCult M3231 medium supplemented with 50 ng/ml Thrombopoietin (TPO; PeproTech, Rocky Hill, NJ), for 7 days according to the manufacturer's protocols (StemCell Technologies, Vancouver, British Columbia, Canada). Colonies containing >3 MKs were counted as CFU–MKs. Duplicate assays were performed for each mouse. At least two mice were analyzed for each sample group.
Mouse FLs, collected on E14.5, were processed into single-cell suspension by successively passing through 18- 21- and 23-gauge needles. Cells were then cultured in DMEM supplemented with 10% FBS, 50 ng/ml murine TPO, 2 mM L-glutamine and penicillin–streptomycin. After 4–5 days, MKs were purified using a discontinuous gradient of bovine serum albumin (BSA, 3%, 1.5%, and 0%, Sigma-Aldrich, St. Louis, MO). About 1–2 × 104 purified MKs were cultured per 6.4 mm diameter well in suspension in flat bottom 96-well plates. After 16 hr of incubation, the fraction of proplatelet-forming MKs per well was scored with a light microscope under label-blinded experimental designs.
For Immunocytofluorescence, FL-derived MKs were allowed to adhere to fibronectin-coated cover slips for 3 hr. Cover slips were rinsed with PBS, fixed with 3.7% formaldehyde, and permeabilized with 0.1% Triton X–100 (Sigma-Aldrich, St. Louis, MO). Cells were blocked with PBS, 2% BSA and incubated with Phalloidin–Rhodamine (gift from Benny Geiger, Weizmann Institute of Science) and anti-alpha–Tubulin antibody (gift from Alexander Bershadsky, Weizmann Institute of Science) for 1 hr. Following blocking, 4'6-Diamidino-2-phenylindole dihydrochloride (DAPI) was added for 5 min before slides were mounted with Immu-Mount (Thermo Scientific). For actin dynamics experiments, MKs were treated after plating for 30 min with 1 mM cytochalasin D (gift from Benny Shilo, Weizmann Institute of Science) and subsequently the drug was washed-out by three medium changes. Cells were fixed with 4% paraformaldehyde at 1 hr or 2 hr after washout and then permeabilized with 0.2% Triton X-100 in PBS. Stress fibers were stained with FITC-conjugated Phalloidin (Sigma-Aldrich, St. Louis, MO) and fluorescent micrographs were captured with a Zeiss LSM510 Laser Scanning confocal microscope.
Flow cytometric analysis was performed on a LSRII flow cytometer (BD Biosciences, San Jose, CA) with FlowJo Version 8.8.7 software (TreeStar, Ashland, OR). BM from 6- to 8-weeks-old mouse femora and tibiae or FL-derived MKs were treated with red blood cell lysis buffer (Ammonium-Chloride-Kalium, ACK, 0.15 M NH4Cl, 0.1 M KHCO3, 1 mM EDTA in PBS). Cells were stained with PE-conjugated anti-CD41 antibody (Abcam, Cambridge, England), FITC-conjugated anti-CD42b (Emfret), or APC-conjugated anti-CD61 antibody. For DNA content analysis, cells were further fixed with 2% paraformaldehyde, stained with 1 µg/ml DAPI, 0.1% BSA (Sigma-Aldrich, St. Louis, MO) and 0.1% Saponin (Sigma-Aldrich, St. Louis, MO). For detailed analysis, freshly obtained BM cells were stained with APC anti-CD150, PE anti-CD41 (Abcam, Cambridge, England), FITC anti-Sca-1, Brilliant Violet anti-CD117, PE-Cy7 anti-CD105, Alexa700 anti-CD16/32, biotin-labeled lineage cell detection cocktail (CD4, CD8α, B220, Ter119, Gr1, CD11b), and streptavidin PerCP–Cy5.5. All antibodies were from BioLegend or eBioscience, unless otherwise indicated. MK-erythroid bipotent progenitors, PreMegE, were gated by lin−c-kit+CD150+CD105−CD41− or lin−sca1−c-kit+CD16/32−, and MK progenitors, MkP, were gated by lin−sca1−c-kit+CD41+ (Pronk et al., 2007; Pronk and Bryder, 2011). Sorted PreMegE and MkP were collected and RNA was extracted using RNeasy micro kit (Qiagen, Venlo, Netherlands).
Quantification of actin intensity and morphocytometry was performed with ImagestreamX flow cytometer and IDEAS 6.0 software (Amnis Corp., Seattle, WA). 2 × 104 FL-derived MKs were stained with APC-conjugated CD61 (Biolegend, San Diego, CA), fixed using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA), and further stained with FITC-conjugated-Phalloidin, Alexa594-conjugated DNaseI (Invitrogen, Life Technologies, Carlsbad, CA) and DAPI. Images were compensated for fluorescent dye overlap by using single-stain controls. Analysis was done on in-focus single cell images as previously described (George et al., 2006) with single cell gating, using the area and aspect ratio features. Cell area was calculated in square microns from brightfield images. Circularity was calculated as average distance of object boundary from center, divided by the variation of this distance. Thus, shapes approximating circle exhibited low variation and gained higher values (in arbitrary units). Actin polarity was calculated using the Delta Centroid XY feature, which calculates the distance (in microns) between image center (brightfield) and the intensity-weighted actin image center (higher values indicate increased polarity). The F/G actin ratio was calculated by dividing the corresponding pixel intensities for each cell.
HEK-293T cells were transfected by calcium phosphate with pLKO.1 encoding shRNAs for knockdown of Wasl, Grlf1, Cfl2, or RFP (TRC, Broad Institute of MIT and Harvard, Cambridge, MA), and lentivirus packaging plasmids (pPAX2 pMD2). Lentivirus supernatants were stored, and for knockdown efficacy assessment, lentiviral particles were added at multiplicity of infection (MOI) of 2, onto 5 × 104 NIH-3T3 cells in 16-mm wells that were incubated with medium containing 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO). Selection of transduced cells was performed with puromycin (2 μg/ml) that was added from the second day and until cells were harvested on day 5.
For MK transduction, approximately 2 × 105 FL cells were cultured up to 4 days in medium supplemented with 50 ng/ml TPO. On the fourth day, cells were purified using a BSA gradient and 1 × 104 cells were placed in 6.4-mm rounded wells in suspension with medium containing 8 μg/ml polybrene that was freshly supplemented with TPO. Lentiviral particles were added at MOI of 25 and transducted through centrifugation at 900×g, 32°C for 90 min. For mock transduction, an equivalent volume of medium was added. Cells were incubated at 37°C overnight, washed in PBS and split into five different 6.4-mm wells in a 96-well flat bottom suspension plate.
Ultra-small core-shell maghemite nanoparticles consisting of a cerium [Ce(III/IV)] cation-doped CAN-γ-Fe2O3 core and a coordinated branched polyethylenimine (b-PEI25) shell (25 kDa) have been prepared according to Israel et al. (patent application PCT/IL2014/050064). Typically, a CAN-γ-Fe2O3 NPs aqueous suspension (1.0 ml, Fe: 1.93 mg/ml–1.93 mg total Fe, 0.0346 mmol Fe, ICP-AES measurement) was diluted in 25.0 ml double distilled water (ddH2O). For the nanoparticle functional shell, we used b-PEI25, which enables electrostatic binding of nucleosides and endosome destabilization by osmotic imbalance, leading to subsequent release of RNA into cell cytoplasm. Therefore, 10.13 mg of polycationic branched b-PEI25, (10.0 mg/ml stock solution, 0.4053 µmol, Sigma-Aldrich, St. Louis, MO) were added to CAN-γ-Fe2O3 NPs at a 1:5.25 ratio (wt/wt). Mild b-PEI25 coordinated coating was accomplished by overnight orbital shaking at room temperature. The resulting crude core-shell b-PEI25-CAN-γ-Fe2O3 nanoparticles were washed three times in 10 ml ddH2O using an Ultra-15 Amicon centrifugal filter (100K, EMD-Millipore, Billerica, MA) operated for 5 min at 4,000 rpm. Then, a size exclusion process was performed by centrifugation (8,000 rpm, 16 min, 18°C and 7,000 rpm, 10 min, 18°C) afforded the corresponding cleaned b-PEI25-CAN-γ-Fe2O3 nanoparticles. Selected nanocomposite characterization of such functional nanoparticles disclosed respective average TEM/DLS NP diameters of 6.86 ± 1.55 and 82.90 nm ± 1.26 (DLS, PDI: 0.195). NP ξ potential (ddH2O) is +31.1 mV. TGA weight loss (N2 atmosphere, 200–410°C temperature range) is 73.62%.
For MK transfection, 0.49 μg (100 nM) of miR-142-3p mimics dsRNA oligonucleotides, or control sequence (Integrated DNA Technologies, Inc., Coralville, IA) were mixed with b-PEI25-CAN-γ-Fe2O3 nanoparticles at a 0.315 Fe/dsRNA wt/wt ratio, incubated 15 min at RT, and then, transfected to 3 × 104 FL-derived MKs in 35-mm plates. mmu-miR-142-3p guide sequence is: U*G*UAGUGUUUCCUACUUUAUmGmGA. An extensively-modified passenger strand sequence is: 5′-C3(spacer)/UmCCmAUmAAmAGmUAmGGmAAmACmACmUAmCA/3′-Cy5.5 (dye). ‘m’ indicates a 2'O-Methyl RNA and ‘*’ indicates a phosphorothioate internucleotide linkage. Cells were then incubated at 37°C overnight, washed in PBS and further cultured in flat bottom 6.4-mm wells in the presence of TPO, for 48 hr after which PPF levels were scored, and RNA was extracted with RNeasy micro kit (Qiagen, Venlo, Netherlands).
Total RNA was isolated with Tri-Reagent (MRC) following manufacturer's instructions. RNA quality was assessed with ND–1000 Nanodrop (Peqlab) and on a 1.5% agarose gel prior to gene-expression profiling using the Mouse Genome Gene 1.0 ST Affymetrix Gene Chip according to the manufacturer's instructions. For real-time Quantitative (q) PCR, cDNA synthesis was carried out by using oligo d(T) primer (Promega) and SuperScript II reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA), following manufacturer's instructions. qPCR analysis of mRNA expression was performed on a LightCycler 480 Real-Time PCR System (F. Hoffmann-La Roche Ltd, Basel, Switzerland), using KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, MA). Efficiency of each primer pair was confirmed by serial dilutions of templates. For quantification of mature mmu–miR–142 forms, cDNA synthesis was carried out by the miScript Reverse Transcription Kit and qPCR reaction utilized miScript SYBR Green PCR Kit (with miScript Universal Primer, Qiagen, Venlo, Netherlands). U6 and hypoxanthine phosphoribosyltransferase 1 (Hprt) were used as a reference for normalization of miRNA and mRNA levels, respectively. All primer sequences are provided in Supplementary file 1.
For protein quantification, FL-derived MKs were lysed in radioimmunoprecipitation (RIPA) buffer with protease and phosphatase inhibitors. Protein concentration was determined using protein assay (Bio-Rad Laboratories Inc. Hercules, CA). 20 μg of proteins were separated by SDS polyacrylamide gel electrophoresis, electrotransferred onto 0.2-mm nitrocellulose membrane, blocked in TBS, 0.1% Tween20 and 5% dry milk for 1 hr and incubated overnight with primary Antibodies: anti-N-WASP/Wasl (4848s; 1:1000; Cell Signaling Technology), anti-Cofilin2 (ab96678; 1:1000; Abcam, Cambridge, England), anti-GAPDH (AM4300; 1:10,000; Ambion) and anti-ATP-synthase (MS507; 1:2000; MitoSciences, Eugene, OR). HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was diluted in TBS, 0.1% Tween20. Immunoreactive proteins were detected using ECL (GE Healthcare, Little Chalfont, UK) and imaged using ImageQuant Las4010. Quantification of blots was performed using ImageJ imaging software.
Microarray data may be found at the Gene Expression Omnibus (GEO) under accession number GSE52141.
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Elaine FuchsReviewing Editor; Rockefeller University, United States
eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
Thank you for sending your work entitled “miR-142 orchestrates a network of actin cytoskeleton cegulators during megakaryopoiesis” for consideration at eLife. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.
The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Elaine Fuchs (Reviewing editor); David Baltimore (peer reviewer). The other reviewer is remaining anonymous.
Your manuscript has now been seen by three experts in the field. As you can see from the reviews included at the end of this email, the comments are largely favorable and all agree that your work is potentially important and well-suited for publication in eLife. They indicate that for the most part, your evidence demonstrates that miR-142 is required for megakaryopoiesis and MK maturation into platelets, and that it functions by modeling cytoskeleton. That said, there are some significant concerns that will need to be addressed prior to moving forward with this paper.
1) Reviewers 1 and 2 raise issues regarding the extent to which you've documented that miR-142-3p is truly responsible for the phenotypes you describe. This seems especially critical given that the knockout was made through a gene trap strategy. Reviewer 1 points out that to argue the point that the observed megakaryopoiesis phenotype is solely due to miR-142‐3p loss, you should demonstrate that by introducing the 3p into the KO, you can fully restore functional MK maturation and platelet biogenesis. Along these lines, it also seems important to at least address whether miR-142b-5p on the other strand is expressed in your cells, whether the gene trap also disrupts its expression, and if so, whether it contributes to the observed megakaryopoiesis phenotype. Finally, reviewer 2 points out that even though you demonstrate the complete loss of miR-142, it is not clear whether the gene trap interferes with e.g. the noncoding RNA that is immediately downstream of the miR-142 coding region. While full rescues in vivo may be beyond the scope of the present study, you should at least confirm the phenotypes and address these points by re-introducing miR-142, 3p, and if relevant, 5p and Brzp1, into your sorted cells for in vitro rescue validations that the phenotypic consequences are indeed due to miR-142-3p loss.
2) Reviewers 2 and 3 persuasively point out the weakness in the data that allows you to conclude that up-regulation of actin-and cytoskeleton-related genes in miR-142 KO cells are responsible for their decreased platelet phenotype. Reviewer 3 suggests relatively straightforward in vitro experiments to bolster your data shown in Figure 5H-I. Reviewer 2 asks for Western quantification in addition to qPCR and luciferase assays, and this seems reasonable. Again, with in vitro studies, which should not take so long to perform, further dissection could be done of how the derepression of actin targets contributes to the observed actin defects. Determining how the dosage affects your competing factors Grlf1/Cfl1 versus Was 1 again will help to discern miR142 functions in this regard. And explanations/discussions of whether the knockdown of your targets truly only show impact in KO and not WT cells are merited.
3) Reviewer 1 suggests that it will be valuable in aiding interpretation to analyze earlier stages of megakaryopoiesis where miR-142 KO MKs already show pronounced defects.
4) On the textual side, Reviewer 2 points out that miR-142 appears to widely express in hematopoietic lineages, and there are several key published papers that indicate important functions of miR-142 in HSC specification and differentiation. These should be discussed as should the issue of whether potential HSC defects could contribute to the observed defects in megakaryocytes. While carrying out experiments to explore this issue seem beyond what is required here, leaving open this possibility allows readers to consider the issue of whether this miRNA has megakaryocyte-specific functions or is more broadly involved in multiple cell types.
This paper titled “miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis” presented solid evidence demonstrating that miR-142 is required for megakaryopoiesis and MK maturation into platelets. Several lines pointed to the mechanism that efficient megakaryopoiesis is achieved through regulating a cohort of actin cytoskeleton regulators. This finding is of significant importance. Most of the conclusions are drawn based on well-presented evidence. However, there are a few points as suggested below need to be further addressed before being considered further for publication in eLife: 1) miR-142b-3p resides on the (+) strand while there is another miRNA that resides within the same locus transcribed from the (-) strand; is miR-142b-5p expressed in this system? Does the gene trap also disrupt its expression? If so, does it contribute to the observed megakaryopoiesis phenotype?
2) The authors show that knocking down of miR-142's targets in miR-142-depleted MKs rescues PPF formation; how about earlier stages of megakaryopoiesis including MkP production and endomitosis where miR-142-/- MKs already reveals pronounced defects?
3) To help argue the point that the observed megakaryopoiesis phenotype is solely due to miR-142-3p loss, the authors should demonstrate that by supplementing miR-142-/- MK with miR-142-3p, they can fully restore functional MK maturation and platelet biogenesis.
In this manuscript, Chapnik et al., describes differentiation defects in a miR-142 KO mouse during megakaryopoiesis. They demonstrated that miR-142, a miRNA highly expressed in hematopoietic lineages, is required for megakaryocyte maturation, in part through regulation of actin dynamics. The work represents an important contribution to understanding miRNA functions in the hematopoietic system and further supports miRNA networks as an important layer of regulation in diverse biological processes. I have several major concerns that should be addressed by the authors.
1) The KO mouse model was generated by a gene-trap strategy instead of a targeted approach. Although the authors demonstrated the complete loss of miR-142, it is not clear whether the insertion of the lacZ cassette interferes with other gene expression e.g. a noncoding RNA Bzrap1 is immediately downstream of the miR-142 coding region. The authors should confirm the phenotypes by re-introducing miR-142 to their sorted cells for at least in vitro validation.
2) miR-142 appears to widely express in hematopoietic lineages. Recent studies from zebrafish indicate important functions of miR-142 in HSC specification and differentiation (Lu X et al, Cell Research 2013; Nimmo et al., Dev Cell 2013). Although the current work focuses on megakaryopoiesis, the authors should at least discuss whether potential HSC defects could contribute to the observed defects in megakaryocytes. In particular, recent clonal analysis of HSCs indicates individual HSCs may contribute differently to distinct lineages (Lu R et al., Nature Biotech 2011). Since miR-142 is broadly expressed in almost all hematopoietic cells, these additional studies could provide critical insights for whether this miRNA has megakaryocyte-specific functions or more broadly involved in multiple cell types.
3) The authors have identified a number of miR-142 targets regulating the actin network through experimental and bioinformatic analyses. For target validation, they should provide Western quantification in addition to qPCR and luciferase assays. Furthermore, although multiple targets are all involved in regulating the actin dynamics, they have divergent functions as pointed out by the authors. For example, Cfl2, Twf1 and Grlf1 function in disassembly of actin filaments whereas Was1 promotes actin polymerization. Thus, it is critical to further dissect how the derepression of these targets contributes to the observed actin defects. Since the authors already knocked down individual targets with shRNA and, surprisingly, observed similar rescue between Grlf1/Cfl1 (disassembly factors) and Was1 (polymerization factor), they should address the issue how the dosage of these competing factors regulated by miR-142 plays important roles in the MK differentiation.
4) miRNA-mediated regulation is often subtle. However, Cfl1 mRNA is upregulated more than 8-fold in the KO (5D) despite the luc assay shows modest regulation (5F). Does the level of Cfl1 mRNA also indirectly regulated by the loss of miR-142?
5) The knockdown of individual targets by shRNA appears to be quite strong (Figure S4) e.g. 70-90% downregulation in the mRNA level. However, the upregulation of these mRNAs in miR-142 is generally modest except for Cfl1 (Figure 5D). Thus, it's surprising that the knockdown of these targets only showed impact in the KO cells but not in the WT cells. I would assume the levels of these genes are probably similarly downregulated by these potent shRNAs in both WT and KO cells. Can the authors provide any further explanation?
In this report, Chapnik et al. used miR-142 deleted mice to explore its role in hematopoiesis and found that it plays a role in platelet development. Accompanying this phenotype, they also reported that the megakaryocyte progenitor population was increased in the miR-142 null mice in the bone marrow and spleen. This effect was attributed to a cell intrinsic mechanism through the use of CD45.1/CD45.2 bone marrow chimera experiments. In complement with additional in vitro experiments, the authors conclude that miR-142 is important for maturation of megakaryocytes. The data presented in the manuscript supports this conclusion. Using genomic approaches, the authors identified a set of actin-related genes that were up-regulated upon miR-142 deletion. They suggest through 3'UTR reporter assays that these genes are direct targets of miR-142. Using RNAi techniques, the authors knocked down the expression of the actin-related genes in miR-142-/- cells and found that it rescued the ability of MKs to generate proplatelets using an in vitro proplatelet formation assay.
Overall, this manuscript is well written and their data in general supports their conclusion. The manuscript reveals a new function of miR-142 and puts miR-142 as a partial regulator of megakaryocytic maturation. However, their conclusion that up-regulation of actin- and cytoskeleton-related genes in miR-142-/- cells is responsible for their decreased platelet phenotype could be strengthened with additional experiments. The ultimate experiments would be to show that RNAi of these genes could increase platelet generation and decrease MkP population in the miR-142-/- mice. However, this requires additional long and laborious experiments. Thus, the authors could alternatively provide further in vitro evidence along with the experiment shown in Figure 5H-I (see comment #8 among the minor points). In summary, there are no major problems with this manuscript.https://doi.org/10.7554/eLife.01964.023
1) Reviewers 1 and 2 raise issues regarding the extent to which you've documented that miR-142-3p is truly responsible for the phenotypes you describe. This seems especially critical given that the knockout was made through a gene trap strategy. Reviewer 1 points out that to argue the point that the observed megakaryopoiesis phenotype is solely due to miR-142‐3p loss, you should demonstrate that by introducing the 3p into the KO, you can fully restore functional MK maturation and platelet biogenesis.
We have reintroduced miR-142-3p into the null model and ameliorated the megakaryocyte (MK) maturity defects, providing persuasive evidence that mature miR-142-3p is responsible for the observed phenotypes reported in our manuscript. The task was a major challenge because primary MKs are notoriously resilient to transfection. After being unsuccessful with commercially-available reagents, we tested a reagent that was recently developed in the labs of Shula Michaeli and Jean-Paul Lellouche at Bar Ilan University. This previously-unpublished reagent was critical for re-introduction of miR-142-3p into MKs and is a robust tool that will be probably utilized by many others in the field. It drove us to add the new tool into the Methods section and 4 additional authors that developed the reagent.
The cellular and molecular signature observed by re-gaining miR-142-3p function is shown in Figures 3 and 5. Figure 3H reports that the re-introduction of miR-142-3p using dsRNA mimetics was sufficient to restore WT PPF levels to miR-142-deficient differentiated MKs. In Figure 5, which focuses on targets of miR-142 upstream of a panel of cytoskeletal regulatory genes, we added a new panel 5E showing that dsRNA overexpression on the background of the genetic null allele recovers the molecular fingerprints of regulation. Thus, re-introduction of miR-142-3p using dsRNA mimetics was sufficient to restore Wasl and Cfl2 expression levels in miR-142-deficient differentiated MKs.
The relevant Results section reads: “Proplatelet formation (PPF) represents the final phase of MK maturation, culminating in platelet release into the bloodstream (Machlus and Italiano, 2013). To analyze whether miR-142 is involved in this process we performed an ex vivo PPF study on FL-derived MKs. Remarkably, we observed a striking 3-fold reduction in miR-142–/– MKs that were extending proplatelets, relative to control MKs (Figure 3G). We next re-introduced miR-142-3p into differentiated MKs, using dsRNA mimetics (Figure 3–figure supplement 1). The introduction of miR-142-3p was sufficient to recapitulate WT PPF levels in cells that are genetically miR-142-deficient (Figure 3H). Conversely, overexpression of miR-142-3p mimics in WT MKs did not yield any significant increase in PPF levels. Thus, miR-142-3p activity is essential for proper MK maturation and its loss results in defective platelet biogenesis.”
Importantly, the new sets of experiments allowed to make a new and important conclusion in the Results section: ”Furthermore, since re-introduction of miR-142-3p into miR-142-/- differentiated MKs restored functional identity, we conclude that there might be a continuous requirement for miR-142-3p activity to maintain MK maturity.” And in the Discussion: “…MKs require sustained miR-142-3p expression, as re-introduction of synthetic miR-142-3p mimetics, even onto differentiated MKs, was sufficient to restore functional maturity.”
Along these lines, it also seems important to at least address whether miR-142b-5p on the other strand is expressed in your cells, whether the gene trap also disrupts its expression, and if so, whether it contributes to the observed megakaryopoiesis phenotype. Finally, reviewer 2 points out that even though you demonstrate the complete loss of miR-142, it is not clear whether the gene trap interferes with e.g. the noncoding RNA that is immediately downstream of the miR-142 coding region. While full rescues in vivo may be beyond the scope of the present study, you should at least confirm the phenotypes and address these points by re-introducing miR-142, 3p, and if relevant, 5p and Brzp1, into your sorted cells for in vitro rescue validations that the phenotypic consequences are indeed due to miR-142-3p loss.
We added data that resolve concerns for both miR-142-5p and Brzp1 in a satisfactory manner. Our measurements reveal that miR-142-5p is expressed hundreds of folds less than miR-142-3p in megakarycyte and practically behaves like a typical ‘passenger’ strand and is discarded during the assembly process of the guide strand (miR-142-3p) into the RISC. See Figure 1B. To the relevant text we added another reference that clearly views miR-142-5p as a passenger (non-functional) strand: “miR-142-3p is the guide strand from the miR-142 hairpin, whereas the sister ‘passenger’ miR-142-5p strand is expressed in negligible levels ((Nimmo et al., 2013) and Figure 1B). The expression of both miR-142-3p and miR-142-5p was abolished in miR-142–/– circulating blood cells as exemplified by quantitative real–time PCR (qPCR, Figure 1B).”
Measurements of Bzrap1, a regulator of synaptic transmission, reveal that it unchanged in Megakaryocytes of miR-142 null mice. See Figure 2–figure supplement 1B. The Results section reads: “We also confirmed that the expression of Bzrap1, a regulator of synaptic transmission (Chardenot et al., 2002; Mittelstaedt and Schoch, 2007) positioned 3.5Kb downstream of miR-142, is unchanged in miR-142–/– MKs (Figure 2–figure supplement 1B).”
2) Reviewers 2 and 3 persuasively point out the weakness in the data that allows you to conclude that up-regulation of actin-and cytoskeleton-related genes in miR-142 KO cells are responsible for their decreased platelet phenotype. Reviewer 3 suggests relatively straightforward in vitro experiments to bolster your data shown in Figure 5H-I. Reviewer 2 asks for Western quantification in addition to qPCR and luciferase assays, and this seems reasonable. Again, with in vitro studies, which should not take so long to perform, further dissection could be done of how the derepression of actin targets contributes to the observed actin defects.
Measuring mRNA levels is an accepted practice in the miRNA field and some leading groups in the field suggest that in fact ‘mammalian microRNAs predominantly act to decrease target mRNA levels’ (Guo et al., 2010 Nature. 466(7308):835-40). However, to comply with the reviewers’ request, we now provide on Figure 5 panels F-G assessment of target protein levels for representative miR-142-3p target genes, which substantiate our analysis. For the Western blot analysis, lysates from WT and miR-142–/– FL–derived MKs were subjected to SDS-polyacrylamide gel electrophoresis. WASL (F) and COFILIN-2 (G) were immunodetected and assessed by densitometry. The results section on page 10 reads: “Western blot analysis revealed upregulation of the protein products of Wasl and Cfl2 in miR-142–/– MKs, further substantiating them as bona fide miR-142-3p targets (Figure 5F-G)”. We were unsuccessful in getting a purchased antibody against mouse Grlf1, another target of miR-142-3p, to work.
Determining how the dosage affects your competing factors Grlf1/Cfl1 versus Was 1 again will help to discern miR142 functions in this regard.
We performed additional experiments to further test the impact of different actin factors, which were described in the original submission. In a new Figure 6 we organized data supporting the claim that miR-142-3p targets a battery of actin cytoskeleton regulators to facilitate proplatelet formation. Panels 6B and 6C reveal the impact of miR-142–/– MKs that were further transduced with shRNAs targeting individual (B), paired (C), or a combined set (B-C) of targets in restoring PPF levels.
As envisioned by the reviewers, more detailed analysis provided additional information. For example: Either Wasl or Cofilin knockdown restored PPF levels in miR-142-deficient MKs (i.e., recovered the wild-type phenotype). However, the knockdown of both targets simultaneously did not rescue the phenotype, presumably because of the opposing activity of Cofilin and Wasl on actin filamentous state. Another observation is that concomitant Cfl2 and Grlf1 knockdown, two proteins that are important for destabilizing actin polymers, enhanced PPF in miR-142 null MKs. The relevant Results section reads: “Additional pairwise target comparison revealed compound relationships between miR-142-3p targets (Figure 6C; Figure 6–figure supplement 2B). Remarkably, knockdown of both Cfl2 and Wasl, which carry out opposing functions within the actin regulatory network, did not yielded any significant increase in PPF levels in miR-142-deficient MKs (Figure 6C). Conversely, concomitant Cfl2 and Grlf1 knockdown, two proteins that are important for destabilizing actin polymers, enhanced PPF in miR-142 null MKs (Figure 6C). Lastly, a pool of shRNA against miR-142-3p targets was able to fully restore PPF capacity to WT levels (Figure 6A-C). These data demonstrate that the expression of a set of actin cytoskeleton regulators should be tightly orchestrated by miR-142-3p in order to effectively promote platelet biogenesis.”
We further discuss how competing factors regulated by miR-142 may play important roles in the MK differentiation, while staying focused and sincere to the main claim of the work: “The actin cytoskeleton participates in a wide array of cellular functions, and the dynamic turnover between its F-actin and monomeric G-actin forms is regulated by a large number of actin-binding proteins. Within this cytoskeletal regulatory network, the set of genes targeted by miR-142-3p contains components with divergent functions. For example, Cfl2 and Twf1 participate in disassembly of actin filaments (Bamburg, 1999; Moseley et al., 2006). Likewise, Grlf1 is a GTPase activating protein that has been implicated in disruption of the organized actin cytoskeleton (McGlade et al., 1993). Wasl, on the other hand, promotes actin polymerization by catalyzing filament branching together with the Arp2/3 complex (Rotty et al., 2013). Thus, miR-142 deficiency destabilizes feedback loops required for actin filament homeostasis, stress fiber formation and actin remodeling. This in turn impairs proplatelet generation and plausibly other MK-intrinsic cellular functions.”
And explanations/discussions of whether the knockdown of your targets truly only show impact in KO and not WT cells are merited.
We discuss why shRNA knockdown of targets impacted the KO cells more than WT cells: “Repression of miR-142-3p actin-associated targets was found to be sufficient in restoring PPF levels in miR-142-deficient MKs. Interestingly, knocking down the same targets in WT MKs did not result in significant increase in PPF capacity over WT baseline levels. This might be related to a certain threshold in PPF levels that cannot be crossed, even when regulation of miR-142-3p is mimicked.”
3) Reviewer 1 suggests that it will be valuable in aiding interpretation to analyze earlier stages of megakaryopoiesis where miR-142 KO MKs already show pronounced defects.
In the revisions we measured several molecular markers of megakaryopoiesis to further characterize the affected steps in megakaryocyte differentiation in bipotent MK–erythroid precursors (PreMegEs) and downstream unipotent MK-progenitors (MkPs). We incorporated these new data into the work in Figure 2 C,D and Results: “To gain insight into the impact of miR-142 nullification on early MK development, we performed a high–resolution flow cytometry assay for the characterization of myeloerythroid progenitors (Pronk and Bryder, 2011; Pronk et al., 2007)… Intriguingly, the expression levels of regulatory markers of MK development […namely Gata1 Gata2, Zfpm1, Klf1, Spi1, Fli1, Runx1, Tal1] remained largely unchanged in miR-142–/– PreMegEs and MkPs, relative to WT controls (Figure 2C,D).”
In the Discussion we state: “miR-142 is dispensable for development of PreMegE, the bi-potent precursors of erythrocytes and MKs and the defects associated with miR-142 deficiency mostly relate to various facets of MK maturity, including increase in cell size, polyploidization and proplatelet elaboration.”
We further clarify in the revised manuscript that actin regulation is probably not the sole pathway downstream of miR-142 and other targets may be involved in regulating MkP proliferation and/or endomitosis. We also state: “It is also likely that additional miR-142 targets participate in the regulation of MK differentiation. For example, the over-representation of miR-142-deficient MkPs might result from a dysregulation of a distinct set of targets that are not necessarily related to actin regulation.”
4) On the textual side, Reviewer 2 points out that miR-142 appears to widely express in hematopoietic lineages, and there are several key published papers that indicate important functions of miR-142 in HSC specification and differentiation. These should be discussed.
We revised the Introduction and Discussion as requested and refer to other recently-published papers related to miR-142 activity in the hematopoietic lineage: “In the present work, we focused on miR-142, a hematopoietic-specific miRNA, which resides in a genomic locus that was previously associated with t(8;17) translocation in B–cell leukemia (Gauwerky et al., 1989). Pioneering experimental evidence has suggested miR-142 involvement in lymphocyte differentiation (Chen et al., 2004) and recently, miR-142 was also shown to play a roles in the specification of definitive hemangioblasts (Lu et al., 2013; Nimmo et al., 2013), and in lymphoid and myeloid lineages (Belz, 2013; Chen et al., 2004; Gauwerky et al., 1989; Huang et al., 2009; Lagrange et al., 2013; Lu et al., 2013; Nimmo et al., 2013; Sonda et al., 2013; Sun et al., 2013; Zhou et al., 2013). Furthermore, miR-142 is involved in compound immune response to North American eastern equine encephalitis virus (Trobaugh et al., 2014) and our work uncovered a key role for miR-142 in the maintenance of CD4+ dendritic cells (Mildner et al., 2013).” And in the Discussion section: “Hematopoietic–specific miR-142 emerges in recent years as a critical regulator of various blood and Lymph cell lineages (Belz, 2013; Chen et al., 2004; Gauwerky et al., 1989; Huang et al., 2009; Lagrange et al., 2013; Lu et al., 2013; Mildner et al., 2013; Nimmo et al., 2013; Sonda et al., 2013; Sun et al., 2013; Zhou et al., 2013)…”
…as should the issue of whether potential HSC defects could contribute to the observed defects in megakaryocytes. While carrying out experiments to explore this issue seem beyond what is required here, leaving open this possibility allows readers to consider the issue of whether this miRNA has megakaryocyte-specific functions or is more broadly involved in multiple cell types.
We performed analysis of earlier developmental stages and conclude that the phenotype emerges downstream of the PreMegE stage (bipotent erythroid-megakaryocyte progenitor) so it is a lineage-restricted phenotype. We come to this conclusion by in depth studies including in vivo competitive repopulation studies and in vitro-differentiated MK culture assays. Furthermore, the expression levels of several regulatory markers of MK development, including Gata1 Gata2, Zfpm1, Klf1, Spi1, Fli1, Runx1, Tal1, all remained unchanged in miR-142–/– PreMegEs. Even downstream at MK progenitors, most markers are unchanged and only Spi1, Fli1 are slightly upregulated (Figure 2C,D). Therefore, we conclude that the phenotype which cannot be depicted at PreMegEs must emerge later in development.
We rephrased the text to better explain our interpretation that miR-142 holds megakaryocyte‐specific functions and is independently involved in other (equally intriguing) facets hematopoiesis and lymphopoiesis: “Our competitive repopulation studies and culture assays reveal that proper miR-142 function is essential, in a cell-intrinsic manner, for differentiation and maturation of MKs independently from additional important roles this miRNA has elsewhere in the hematopoietic system.”https://doi.org/10.7554/eLife.01964.024
- Alexander Mildner
- Steffen Jung
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Steffen Jung
- Steffen Jung
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
- Eran Hornstein
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We are grateful to G Damari and R Haffner-Krausz for transgenic mouse production and to O Higfa and Y Melamed for excellent mouse husbandry. Y Lotem, N Pencovich, O Ben-Ami, E Geron, B Shilo, E Schejter, A Sharp, R Straussman, and E Ariel contributed valuable know-how and protocols. K Buchman contributed to patent application PCT/IL2014/050064. M Pick, Y Groner and M de Brujin contributed insightful discussions, D Aronowitz performed English editing.
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol of the Weizmann Instituter of Science. The protocol, entitled ‘miR-142 in hematopoietic lineage development’ was approved under Permit Numbers: 02930513-3 and 00350111-1. Every effort was made to minimize suffering.
- Elaine Fuchs, Rockefeller University, United States
© 2014, Chapnik et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.