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Dissecting the roles of miR-140 and its host gene

Nature Cell Biology volume 20, pages 516–518 (2018)Cite this article

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To explore these two possibilities, we took advantage of the CRISPR–Cas9 system to generate miR-140 and Wwp2 KO mice, and miR-140/Wwp2 double-KO mice. Single-guide (sg)RNAs, which target the second exon of the Wwp2 gene and/or the miR-140-5p sequence, were microinjected into mouse zygotes together with hCas9 mRNA (see Methods for the details). As a result, we isolated the alleles with small insertions or deletions (indels) in the Wwp2 locus (Wwp21ins; miR-140+), the miR-140 locus (Wwp2+; miR-14014del), or in both loci (Wwp21del; miR-140132del) (Fig. 1b,c). Western blot and quantitative polymerase chain reaction (qPCR) analyses confirmed that no Wwp2 protein was detected in the chondrocytes of mice with mutated Wwp2 alleles (Fig. 1e) and mature miR-140 was absent in chondrocytes of mice with mutated miR-140 alleles (Fig. 1d). From this point, miR-140 KO refers to Wwp2+/+; miR-14014del/14del, Wwp2 KO and miR-140/Wwp2 double-KO refer to Wwp21ins/1ins; miR-140+/+ and Wwp21del/1del; miR-140132del/132del, respectively.

Next, we examined the craniofacial phenotype of these mice. The skulls of 30-day-old mice were observed with micro-computed topography (CT) (Fig. 1f). First, we confirmed that the skulls of miR-140 KO mice were truncated as in the previous report (Fig. 1f). However, to our surprise, the skulls of Wwp2 KO mice were undistinguishable from that of wild-type (WT) mice (Fig. 1f). The miR-140/Wwp2 double-KO mice had truncated skulls, but no apparent difference was seen when compared with the miR-140 KO skull (Fig. 1f). These results suggest that, at least in our system, the loss of Wwp2 protein alone does not cause the craniofacial truncation phenotype. Furthermore, simultaneous depletion of Wwp2 protein and miR-140 does not promote the craniofacial phenotype caused by loss of miR-140 (Fig. 1f). In summary, our results suggest that skull truncation correlates with loss of miR-140 expression, but not with Wwp2 protein level, and that the functional coherence between these two factors does not take place during craniofacial skeletal development. Our results also suggested that the craniofacial phenotype observed in Wwp2GT/GT mice was not due to the loss of Wwp2 protein, but to the unintentional loss of miR-140 expression by the gene-trap cassette. Consistent with this idea, not only Wwp2 protein but also miR-140 are significantly reduced with the Wwp2GT allele (Fig. 2b,c). To further elucidate if loss of Wwp2 protein or miR-140 is responsible for the craniofacial phenotype of Wwp2GT/ GT mice, we took advantage of a compound heterozygous strategy: we crossed Wwp2GT/GT mice with our miR-140 KO or Wwp2 KO mice and examined the correlation between craniofacial phenotype and expression level of miR-140 and Wwp2 protein (Fig. 2a). Subsequently, while compound heterozygotes of Wwp2GT and Wwp2 KO alleles (Wwp2GT/1ins; miR-140GT/+) showed no craniofacial phenotype with no Wwp2 protein and reduced but significant miR-140 expression (Fig. 2b–d), compound heterozygotes of Wwp2GT and miR-140 KO (Wwp2GT/+; miR-140GT/14del) showed clear craniofacial truncation with reduced but significant Wwp2 protein and no miR-140 expression (Fig. 2b–d). These results demonstrated that craniofacial truncation appeared when expression of miR-140, but not Wwp2, was depleted from the allele combined with Wwp2GT. From these results, we concluded that miR-140, not Wwp2 protein is required for proper craniofacial development.

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Fig. 1: Generation and craniofacial phenotype of miR-140, Wwp2 and miR-140; Wwp2 double KO mice.
Fig. 2: Compound heterozygous analysis between WWP2GT allele and miR-140 KO or Wwp2 KO allele.

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Acknowledgements

We thank Koichi Masuda (UCSD) for the kind cooperation in microCT analysis. This research is supported by AMED-CREST from AMED (JP15gm0410001, JP17gm0810008), Daiichi Sankyo Foundation of Life Science, JSPS KAKENHI [Grant Number: 26113008, 15H02560, 15K15544], and grants from the National Institutes of Health [Grant Number: AR050631, AR065379] to H.A., the Grant from the National Center for Child Health and Development (Grant Number 25-1), the Uehara memorial foundation, the Nakajima Foundation and JSPS KAKENHI (16K18558) for M.I.

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  1. These authors contributed equally: Masafumi Inui and Sho Mokuda.

Authors and Affiliations

  1. Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan

    Masafumi Inui, Tempei Sato, Moe Tamano, Shuji Takada & Hiroshi Asahara

  2. Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa, Japan

    Masafumi Inui

  3. Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA

    Sho Mokuda & Hiroshi Asahara

  4. Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    Tempei Sato & Hiroshi Asahara

  5. AMED-CREST, Japan Agency for Medical Research and Development (AMED), Tokyo, Japan

    Hiroshi Asahara

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Contributions

M.I., S.M. and T.S. analysed the KO mice, M.T. performed the microinjections. M.I., S.T. and H.A. planned the experiments and wrote the manuscript.

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Correspondence to Masafumi Inui or Hiroshi Asahara.

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Inui, M., Mokuda, S., Sato, T. et al. Dissecting the roles of miR-140 and its host gene. Nat Cell Biol 20, 516–518 (2018). https://doi.org/10.1038/s41556-018-0077-4

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