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. 2014 Jun 30;9(6):e101153.
doi: 10.1371/journal.pone.0101153. eCollection 2014.

Phenotypic characterization of miR-92a-/- mice reveals an important function of miR-92a in skeletal development

Daniela Penzkofer  1 , Angelika Bonauer  1 , Ariane Fischer  1 , Alexander Tups  2 , Ralf P Brandes  3 , Andreas M Zeiher  4 , Stefanie Dimmeler  5
Affiliations

Phenotypic characterization of miR-92a-/- mice reveals an important function of miR-92a in skeletal development

Daniela Penzkofer et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MiR-92a−/− mice survive at a reduced Mendelian ratio.
(A) Representative agarose gel picture of PCR products (WT allele: 1718 bp; miR-92a knockout (KO) allele: 422 bp) for genotyping of WT, miR-92a+/− and miR-92a−/− mice. (B) MiR-92a expression in the heart of WT, miR-92a+/− and miR-92a−/− mice. (C) Expression of the miR-17∼92 cluster members miR-17, miR-18a, miR-19a, miR-20a and miR-19b in the heart of WT, miR-92a+/− and miR-92a−/− mice. (D) Observed as well as by Mendelian ratios predicted percentage of weaned WT, miR-92a+/− and miR-92a−/− mice derived from mating of miR-92a+/− mice. Data are represented as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA; # P<0.05 by Clopper-Pearson interval, $ P<0.01 by chi-square test.
Figure 2
Figure 2. MiR-92a deficiency does not induce obvious defects in the hematopoietic system.
Fraction of B220+CD43+ pro-B cells and B220+CD43− pre-B cells measured in blood (A) and bone marrow (B) of male WT, miR-92a+/− and miR-92a−/− mice. CD45 (C, D), CD19 (E, F), IgM (G, H) positive cells measured in blood and bone marrow of WT, miR-92a+/− and miR-92a−/− mice. Data are represented as mean ± SEM.
Figure 3
Figure 3. MiR-92a−/− mice exhibit a reduced body weight.
Body weight of male juvenile (A) and adult (B) WT, miR-92a+/− and miR-92a−/− mice. (C) Weight of heart, lung, liver, spleen, and kidney of adult male WT, miR-92a+/− and miR-92a−/− mice either as raw data or normalized to body weight. (D) Weight of WT, miR-92a+/− and miR-92a−/− E15.5 embryos. Data are represented as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA in A and B, by student’s t-test in C.
Figure 4
Figure 4. Constitutive deletion of miR-92a in mice causes skeletal defects.
(A) Body length of WT and miR-92a−/− mice measured from the tip of the nose to the basis of the tail on the animals itself. (B) Length of dissected tibias of WT and miR-92a−/− mice. (C) Representative pictures of whole body µCT-scans of WT and miR-92a−/− mice, scale bar: 1 cm. Head length (D) and ratio of mesophalanx V/metacarpale V of the forelimbs (E) measured on µCT-scans of WT and miR-92a−/− mice. (F) Representative pictures of µCT-scans of forelimbs from WT and miR-92a−/− mice, mesophalanx V and metacarpale V are indicated. (G) Ratio of middle phalanx V/metatarsal V of the hind limbs measured on µCT-scans of WT and miR-92a−/− mice. (H) Bone density of WT and miR-92a−/− mice measured by dual energy X-ray absorptiometry. Data are represented as mean ± SEM, **P<0.01, ***P<0.001 by student’s t-test.
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Grants and funding

The study was supported by the European Union (ERC advanced grant “AngiomiR”). The µCT core facility is supported by the German Center of Cardiovascular Research (BMBF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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