No abstract

Summary MicroRNAs (miRNA) are small, non-coding RNAs that regulate gene expression post-transcriptionally. We investigated the hypothesis that Bone Morphogenetic Protein (Bmp)-signaling regulates miRNAs in cardiac progenitor cells. Bmp2 and Bmp4 regulate OFT myocardial differentiation via regulation of the miRNA 17-92 cluster. In Bmp mutant embryos, myocardial differentiation was delayed and multiple miRNAs encoded by miRNA 17-92 were reduced. We uncovered functional miRNA17-92 seed sequences within the 3′ UTR of cardiac progenitor genes such as Isl1 and Tbx1. In both Bmp and miRNA 17-92 mutant embryos, Isl1and Tbx1 expression failed to be correctly downregulated. Transfection experiments indicated that miRNA 17 and miRNA 20a directly repressed Isl1and Tbx1. Genetic interaction studies uncovered a synergistic interaction between miRNA 17-92 cluster and Bmp4 providing direct in vivo evidence for the Bmp-miRNA 17-92 regulatory pathway. Our findings indicate that Bmp-signaling directly regulates a miRNA-mediated effector mechanism that downregulates cardiac progenitor genes and enhances myocardial differentiation.

IntroductionErythropoiesis is the complex process during which a fraction of primitive multipotent hematopoietic stem cells become committed to the red cell lineage, undergoing erythroid progenitors (burstforming unit erythroid [BFU-E] and colony-forming unit erythroid [CFU-E]), normoblasts, erythroblasts, reticulocytes, and ultimately differentiating to mature erythrocytes. 1 This process is regulated by various factors such as erythropoietin, testosterone, estrogen, interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-9. 2 Activin, a member of the transforming growth factor-␤ (TGF-␤) superfamily, plays an important role in modulating proliferation and differentiation of erythroid progenitors. [2][3][4] In cooperation with erythropoietin, activin promotes erythroid differentiation by increasing the number of hemoglobin-synthesizing colonies derived from the progenitors CFU-E and enhances the formation of BFU-E. 3,[5][6][7] Activin, like other TGF-␤ superfamily members, signals through 2 types of transmembrane receptors that have intrinsic serine/ threonine kinase activity. 8,9 It binds to type II activin receptor, leading to the recruitment, phosphorylation, and activation of type I activin receptor (ALK4, also known as ActRIB). The activated ALK4 transiently interacts with and then phosphorylates Smad2 and Smad3, which, upon phosphorylation, form a heterocomplex with Co-Smad (Smad4), and the resulting Smad complex is accumulated in the nucleus, binds to the promoter of the target genes, and regulates their expression. 10,11 MicroRNAs (miRNAs) are 21-to 25-nucleotide small regulatory RNAs that modulate gene expression by targeting mRNA for degradation or blocking translation via base-pairing to complementary sites in the 3Ј-untranslated region (3Ј-UTR) of the target mRNAs. 12,13 Hundreds of miRNAs have been identified and they participate in a diverse collection of regulatory events. Investigation of the miRNA expression profile in the course of hematopoietic development suggests their potential regulatory roles in hematopoietic differentiation. 14,15 For example, ectopic expression of miR-181 in hematopoietic progenitor cells increased the proportion of B-lineage cells, whereas miR-142s and miR-223 promote the differentiation to T-lineage but not to B-lineage or myeloid cells. 15 MiR-155 transgenic mice exhibited pre-B cell proliferation and B cell malignancy. 16 Overexpression of miR-150 in hematopoietic stem cells specifically impaired the formation of mature B cells by blocking the transition from the pro-B to the pre-B stage. 17 Although many miRNAs have been implicated in hematopoiesis, few of their targets have been identified. MiR-221 and miR-222, both of which are clustered on the X chromosome and were suggested to inhibit normal erythropoiesis and erythroleukemic cell growth, down-modulated kit receptor expression. 18 In this study, we showed that miR-24 could decrease human ALK4 (hALK4) expression at the mRNA and the protein levels through binding to the 3Ј-untranslated r...