TY - JOUR
T1 - Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions
AU - Ota, Hiromitsu
AU - Ito-Matsuoka, Yumi
AU - Matsui, Yasuhisa
N1 - Funding Information:
We would like to thank Dr. Takashi Shinohara for sharing GS cells, Asuka Takehara for maintenance of mouse colonies, all the members of the Cell Resource Center for Biomedical Research for helpful discussions, the Center of Research Instruments in the Institute of Development, Aging, and Cancer and Biomedical Research Unit of Tohoku University Hospital for use of instruments and technical support.
Funding Information:
Funding:H.O.wassupportedbyaGrant-in-Aid (KAKENHI)forYoungScientists(B)(grant #JP16K20908)fromtheMinistryofEducation, Culture,Sports,ScienceandTechnologyofJapan (http://www.mext.go.jp/en/index.htm).Y.M.was
Publisher Copyright:
© 2019 Ota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2019/2
Y1 - 2019/2
N2 - MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miRNAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/ Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogenesis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 30-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in spermatogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development.
AB - MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miRNAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/ Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogenesis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 30-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in spermatogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development.
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U2 - 10.1371/journal.pone.0211739
DO - 10.1371/journal.pone.0211739
M3 - Article
C2 - 30707741
AN - SCOPUS:85060953837
SN - 1932-6203
VL - 14
JO - PLoS ONE
JF - PLoS ONE
IS - 2
M1 - e0211739
ER -