miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis
- Weizmann Institute of Science, Israel
- Cancer Research Center, Sheba Medical Center, Israel
- Hadassah Medical Center, Israel
- Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Israel
- Bar-Ilan University, Israel
- Tel Aviv University, Israel
Figures
Figure 1 with 1 supplement
Pronounced thrombocytopenia in miR-142−/− mice.
(A) Left panel: schematic representation of the gene trap cassette targeting the murine miR-142 locus. The WT and mutant loci are shown with the gene trap-targeting vector. pre-miR-142 is shown as a …
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Figure 1—source data 1
Mendelian distribution of miR-142 intercrosses.
Genotypes of E14.5 and P21 offspring from miR-142+/− intercrosses reveals partial perinatal or juvenile lethality in miR-142−/− mice. The actual and expected number of mice for each genotype at the indicated stages is shown.
- https://doi.org/10.7554/eLife.01964.004
Figure 1—figure supplement 1
miR-142 hematopoietic-intrinsic expression is required for thrombopoiesis.
Schematic diagram of the experimental design for re-introduction of miR-142−/− BM into lethally irradiated WT mouse recipient (left panel), which results in reduced platelet counts relative to …
Figure 2 with 2 supplements
Perturbed myeloerythroid development in the absence of miR-142.
(A) Diagram of gating strategy used to define the myeloerythroid progenitor populations (top panel), and representative FACS profiles of mutant miR-142−/− and WT BM cells (bottom panels). (B) Flow …
Figure 2—figure supplement 1
Gene expression in miR-142−/− MKs.
(A and B) qPCR expression analyses confirm nullification of miR-142-3p and miR-142-5p (A) and similar Bzrap1 expression (B) in miR-142−/− MKs, relative to WT controls. Representative results from …
Figure 2—figure supplement 2
Splenomegaly and increased numbers of splenic MKs in miR-142−/− mice.
(A) Representative spleen sections were stained with hematoxylin and eosin (H&E, top panel) and anti-von Willebrand Factor (anti vWF; bottom panel). Arrows indicate MKs. Scale bars, 100 μm. (B) A …
Figure 3 with 1 supplement
Impaired maturation of miR-142−/− MKs.
(A) Representative BM sections of miR-142−/− vs WT controls, stained with anti-von Willebrand Factor (anti vWF). In the bottom right corner of each section there is an enlarged image of a …
Figure 3—figure supplement 1
miR-142-3p expression in differentiated MKs following transfection of dsRNA mimetics. qPCR expression analyses confirm overexpression of miR-142-3p in WT and miR-142−/− FL-derived MKs transfected with miR-142-3p mimic, relative to MKs transfected with control mimics.
**p<0.005.
Figure 4
Disturbed actin cytoskeletal architecture and dynamics in the absence of miR-142.
(A) Representative micrographs of WT and miR-142−/− FL-derived MKs, cultured for 5 days with TPO and subsequently plated on fibronectin-coated cover-slips for 3 hr. F-actin (phalloidin, red), …
Figure 5 with 4 supplements
miR-142 Regulates a group of cytoskeletal regulatory genes during megakaryopoiesis.
(A) A log2-scale scatter plot, presenting the expression of mRNAs from FL-derived WT MKs (x axis), and miR-142−/− MKs (y axis). The inset table depicts the number of genes that are significantly up- …
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Figure 5—source data 1
GO analysis for differentially regulated genes (>twofold) in miR-142−/− MKs.
- https://doi.org/10.7554/eLife.01964.013
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Figure 5—source data 2
Genes commonly up-regulated (>1.5-fold) in miR-142−/− MKs and DCs (CD24+ and CD24−).
TargetScan predicted targets of miR-142-3p miR-142-5p are indicated.
- https://doi.org/10.7554/eLife.01964.014
Figure 5—figure supplement 1
Expression distribution plot of miR-142 putative targets.
miR-142-3p TargetScan-predicted targets (blue right panel) are over-represented among genes upregulated in miR-142−/− FL-derived MKs (p<10E−16). miR-142-5p TargetScan-predicted targets (blue, left …
Figure 5—figure supplement 2
qPCR expression analysis of miR-142 putative targets in precursor cell populations.
qPCR expression analysis of Wasl and Cfl2 in PreMegE (left panel) and MkP (right panel) precursor cell populations sorted from miR-142−/− and WT BMs. *p<0.05; ***p<0.0005.
Figure 5—figure supplement 3
miR-142-3p directly regulates cytoskeletal genes.
Schematic representations of 3′UTRs of cytoskeletal genes (A) Cfl2, (B) Wasl, (C) Bod1, Twf1, (E) Itgav, (F) Grfl1 with miR-142-3p binding sites and the corresponding mutations made to test direct …
Figure 5—figure supplement 4
miR-142-3p directly regulates cytoskeletal genes.
Relative luciferase activity of reporters that harbor the 3′UTR of novel miR-142-3p targets: Bod1 (A), Twf1 (B), and Itgav (C). Luciferase reporter activity is repressed by introduction of miR-142 …
Figure 6 with 2 supplements
miR-142-3p targets a battery of actin cytoskeleton regulators to facilitate proplatelet formation.
(A) Representative micrographs of WT and miR-142−/− (KO) FL-derived MKs, transduced with the indicated shRNA vectors and cultured for 48 hr with TPO. White arrows denote MKs extending proplatelets. …
Figure 6—figure supplement 1
Knockdown validation of miR-142-3p targets.
qPCR expression analysis of Cfl2, Wasl, and Grlf1 genes in NIH-3T3 fibroblasts that were transduced with either lentivrial vectors encoding shRNAs directed against miR-142-3p targets (shCfl2, …
Figure 6—figure supplement 2
Knockdown of miR-142-3p targets in WT MKs has no effect on proplatelet formation.
WT MKs transduced with shRNAs targeting miR-142-3p targets exhibited PPF levels slightly higher than WT MKs that were transduced with a control shRNA-targeting RFP. Representative results from one …
Additional files
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Supplementary file 1
Sequences of primers used in this study.
- https://doi.org/10.7554/eLife.01964.022
