miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis

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 This previously-unpublished reagent was critical for re-introduction of miR-142-3p into MKs and is a robust tool that will be probably utilized by many others in the field.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.”