Bmp signaling regulates myocardial differentiation from cardiac progenitors through a MicroRNA-mediated mechanism

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. 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 miRNA-17-92 seed sequences within the 3' UTR of cardiac progenitor genes such as Isl1 and Tbx1. In both Bmp and miRNA-17-92 mutant embryos, Isl1 and Tbx1 expression failed to be correctly downregulated. Transfection experiments indicated that miRNA-17 and miRNA-20a directly repressed Isl1 and 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.

Figure 1. Myocardial differentiation in Bmp mutant embryos

(A-J) Immunofluorescence with MF20 (green, denoted by arrow). Genotypes are shown. (K) Myocardial thickness at E10.5. (L,M) Bar graph for QRTPCR assay. (N-S) Immunofluorescence with Isl1 antibody (green, arrows) at E9.75. (T-W) Whole mount in situ hybridization at E9.5. ba, branchial arch; e, eye. Nuclei stained with TO-PRO-3 iodide (TP3) (red) in all Immunofluorescence. *, (P < 0.05; error bars represent SEM).

Figure 2. Bmp-signaling regulates miRNAs

(A) miRNA array from Bmp2/4 CKO embryos. MiRNAs from the miR-17-92 complex and homologous complexes (arrows). Other cardiovascular miRNAs (asterisk). (B) QRTPCR validation of miRNA array data at E9.5. *, statistically significant difference (P < 0.05). (C) Genomic structure for miR-17-92 and two orthologous clusters (left) and sequences of mature miRNA sequences of miR-17 family with seed sequence yellow highlighted (right) (Modified from Mendell 2008). (D-I) In situ hybridization: (D) miR-17 is expressed in OFT (upper arrow) and SHF (lower arrow) at E9.5. (E) Blank control for E9.5 miR-17 in situ hybridization. (F, H) pri-miR-17-92 in situ whole mount (F) and section (H) at E10.5. Proximal OFT (arrow). (G, I) Sense control for pri-miR-17-92 in situ. ba, branchial arch; oft, outflow tract. (J-L) MiR-17-92 5′ upstream Luciferase reporter (miR-17-92-pGL3) activity co-transfected with constitutively active Alk3 (caAlk3) (J), pcDNA3.1-Smad6 (K), and pSR siSmad1 (L). *, (P <0.05; error bars represent SEM). (M) In vivo chromatin immunoprecipitation (ChIP) using E9.5 hearts with indicated antibodies to IP chromatin fragment containing the miR-17-92 5′ upstream Bmp/Smad element.

Figure 3. MiR-17-92 cardiac phenotype and Bmp4 genetic interaction

(A-D) Real time qPCR analysis. *, statistically significant difference (P < 0.05). (E) Immunofluorescence at E9.5. Boxed areas in c, i, o are correspondingly shown at higher magnification in d-f, j-l and p-r. Tangent line with branchial arch landmark (dashed line), reveals Isl1-expressing progenitors toward proximal OFT (left of dashed line) were persistent in miR-17-92 null mutants (arrows in h, i, k and l) while decreased in controls (arrows in b, c, e and f) and almost extinguished in miR-17-92 OE mutants (arrows in n, o, q and r). Persistent Isl1-expressing progenitors in endocardium of miR-17-92 null mutants (arrowheads in h, i, k and l). ao, aorta; la, left atrium; ra, right atrium; pt, pulmonary trunk. (F-K) Whole mount (F, H, J) and sections (G, I, K) of embryos with the indicated genotypes and stages. Arrows in G, I, K denote the proximal cushion mesenchyme and arrowhead in K the OFT septum. (L-N) E14.5 embryos showing normal and defective OFT alignment (arrows). (O, P, Q) Transverse sections through OFT showing normal and defective separation between the aorta and pulmonary trunk (arrows). (R, S) Real time qPCR analysis. *, (P < 0.05; error bars represent SEM).

Figure 4. Isl1 and Tbx1 are miR-17-92 targets

(A-C) Phylogenetic sequence alignment and mutagenesis of miR-17-92 family seed sequence in Isl1 3′ UTR (A) and Tbx1 3′ UTR (B, C). (D-K) Luciferase reporter assays with reporters and miR's as labelled. *, (P < 0.05; error bars represent SEM).