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MicroRNAs (miRNAs, miRs) emerged as key regulators of gene expression. Germline hemizygous deletion of the gene that encodes the miR-17~92 miRNA cluster was associated with microcephaly, short stature and digital abnormalities in humans. Mice deficient for the miR-17~92 cluster phenocopy several features such as growth and skeletal development defects and exhibit impaired B cell development. However, the individual contribution of miR-17~92 cluster members to this phenotype is unknown. Here we show that germline deletion of miR-92a in mice is not affecting heart development and does not reduce circulating or bone marrow-derived hematopoietic cells, but induces skeletal defects. MiR-92a−/− mice are born at a reduced Mendelian ratio, but surviving mice are viable and fertile. However, body weight of miR-92a−/− mice was reduced during embryonic and postnatal development and adulthood. A significantly reduced body and skull length was observed in miR-92a−/− mice compared to wild type littermates. µCT analysis revealed that the length of the 5th mesophalanx to 5th metacarpal bone of the forelimbs was significantly reduced, but bones of the hindlimbs were not altered. Bone density was not affected. These findings demonstrate that deletion of miR-92a is sufficient to induce a developmental skeletal defect.
Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. Wnt proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of Wnts on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6–10 days after co-culture, cardiac differentiation was determined by α-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with Wnt11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7 ± 0.3-fold), whereas Wnt3A-conditioned medium showed no effect. Cell fusion was not affected by Wnt11-conditioned medium. Because Wnts inhibit glycogen synthase kinase-3β, we further determined whether the glycogen synthase kinase-3β inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mm) did not affect CPC differentiation. In contrast, Wnt11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol 12-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with Wnt11-conditioned medium. Our data demonstrate that Wnt11, but not Wnt3A, augments cardiomyogenic differentiation of human CPCs. Wnt11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.