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Herpesvirus Saimiri Noncoding RNA Demián Cazalla, Et Al

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Herpesvirus Saimiri Noncoding RNA Demián Cazalla, Et Al Down-Regulation of a Host MicroRNA by a Herpesvirus saimiri Noncoding RNA Demián Cazalla, et al. Science 328, 1563 (2010); DOI: 10.1126/science.1187197 This copy is for your personal, non-commercial use only. If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this infomation is current as of December 1, 2010 ): A correction has been published for this article at: http://www.sciencemag.org/content/329/5998/1467.3.full.html Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/328/5985/1563.full.html Supporting Online Material can be found at: on December 1, 2010 http://www.sciencemag.org/content/suppl/2010/06/15/328.5985.1563.DC1.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/328/5985/1563.full.html#related This article cites 24 articles, 16 of which can be accessed free: http://www.sciencemag.org/content/328/5985/1563.full.html#ref-list-1 This article has been cited by 1 articles hosted by HighWire Press; see: www.sciencemag.org http://www.sciencemag.org/content/328/5985/1563.full.html#related-urls This article appears in the following subject collections: Molecular Biology http://www.sciencemag.org/cgi/collection/molec_biol Downloaded from Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2010 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS. REPORTS To analyze the cell cycles of germ cells in the To confirm that the nos2-expressing Gs cells 6. K. Eggan, S. Jurga, R. Gosden, I. M. Min, A. J. Wagers, germinal cradles, we assessed the S-phase index, a generated fertile eggs, F1 embryos from heat- Nature 441, 1109 (2006). 7. R. R. Tokarz, in The Vertebrate Ovary, R. E. Jones, Ed. measure of the percentage of nuclei in the S phase. treated transgenic medaka were genotyped (fig. S8 (Plenum, New York, 1978), pp. 145–179. Adult female medaka were exposed to BrdU for and table S2). Some embryos possessed an un- 8. R. A. Wallace, K. Selman, J. Electron Microsc. Tech. 16, various periods of time (fig. S6A). After 24 hours, cleaved loxP region, whereas others exhibited the 175 (1990). nearly 60% of the Gs cells were BrdU positive, characteristic pattern of successful loxP-mediated 9. C. Morinaga et al., Proc. Natl. Acad. Sci. U.S.A. 104, 9691 (2007). and the rate of newly stained cells slowed over the recombination (Fig. 2E). Embryos with the cleaved 10. V. P. I. Vidal, M. C. Chaboissier, D. G. de Rooij, A. Schedl, following week such that about 75% of the Gs loxP region were produced for 3 months after Nat. Genet. 28, 216 (2001). cells were BrdU positive. Thus, Gs cells appear heat treatment (table S2), indicating that the pop- 11. T. Wagner et al., Cell 79, 1111 (1994). to be heterogeneous. Piecewise linear regression ulation of nos2-expressing Gs cells was capable 12. N. Klüver, M. Kondo, A. Herpin, H. Mitani, M. Schartl, analysis revealed two distinct populations: fast- of continuously generating fertile eggs. Dev. Genes Evol. 215, 297 (2005). 13. S. Nakamura et al., Mol. Reprod. Dev. 75, 472 (2008). dividing (Gsf) and slow-dividing (Gss) germ cells. We identified and characterized ovarian cords 14. H. Kurokawa et al., Dev. Growth Differ. 48, 209 (2006). BrdU pulse-chase experiments also identified within the germinal epithelia of medaka ovaries. 15. M. Tanaka, M. Kinoshita, D. Kobayashi, Y. Nagahama, label-retaining germ cells among the Gs cells (fig. These cords were composed of sox9b-expressing Proc. Natl. Acad. Sci. U.S.A. 98, 2544 (2001). S6, B to D). These experiments suggest that at least cells and contained mitotic nos2-expressing Gs 16. Y. Aoki et al., Dev. Dyn. 237, 800 (2008). 17. Y. Aoki, S. Nakamura, Y. Ishikawa, M. Tanaka, Zoolog. 60% of Gs cells are Gsf cells. In contrast, the per- cells in discrete structures referred to as germinal Sci. 26, 112 (2009). centage of BrdU-positive Gcys nuclei increased cradles. These mitotic oogonia continually gave rise 18. T. Iwai et al., Exp. Cell Res. 312, 2528 (2006). from about 35% after 1 hour of BrdU labeling to to germ cells that developed in the ovary, finally 19. E. Passegué, A. J. Wagers, S. Giuriato, W. C. Anderson, nearly 100% after 24 hours. Thus, no quiescent resulting in fertile eggs. Thus, we conclude that the I. L. Weissman, J. Exp. Med. 202, 1599 (2005). 20. Y. Stahl, R. Simon, Int. J. Dev. Biol. 49, 479 (2005). Gcys cells were present in the germinal cradles. ovarian cord harbors the histological niche within 21. J. W. Zhang et al., Nature 425, 836 (2003). We next addressed the potential functions of the ovary where germinal cradles are formed 22. We thank M. Yamashita and T. Iwai for anti-medaka SYCP the Gs cells. We conducted experiments to de- (Fig. 3). Moreover, these cradles contain oogonia antibodies, T. Czerny for plasmids containing the hs termine whether Gs cells facilitate the recovery characteristic of germline stem cells that contrib- promoter region, and S. Yoshida for helpful discussions. We are grateful to Y. Ichikawa and C. Kinoshita for of Gcys cells after busulfan treatment. Three- to ute to the production of fertile eggs (Fig. 3). maintaining the fish colony. This work was supported in 4-month-old female medaka were treated with The germinal cradle in the ovarian cord is rem- part by Grants-in-Aid for Scientific Research on 10 ng/ml busulfan for 1 week to eliminate mitot- iniscent of the germarium from the Drosophila Innovative Areas, “Gamete Stem Cells” (grant 21116509) ically active Gcys cells. The number of germinal ovary (1); both promote the development of germ and for Young Scientists (B) (21770072) (to S.N.), and on December 1, 2010 cradles with Gcys cells was markedly reduced cells from germline stem cells to very early diplo- for Scientific Research on Priority Areas (B) (21370101); the National BioResource Project Medaka; and the Daiko 1 month after treatment, before recovering to the tene oocytes in a unique histological compartment Foundation and the Center for the Promotion of numbers observed in untreated control samples within the ovary. These similarities might reflect Integrated Science (CPIS) of SOKENDAI (to M.T.). 3 months after the treatment (fig. S7). These re- a fundamental process governing oogenesis across Supporting Online Material sults suggest that the Gs cells present 1 month animal species. www.sciencemag.org/cgi/content/full/science.1185473/DC1 after oogenesis was disrupted are capable of re- Materials and Methods generating Gcys cells in the germinal cradles. References and Notes SOM Text Figs. S1 to S9 To confirm that Gs cells in the ovary were stem 1. M. D. Wong, Z. Jin, T. Xie, Annu. Rev. Genet. 39, 173 Tables S1 and S2 cell–like germ cells, we conducted clonal analysis (2005). References www.sciencemag.org 2. T. C. A. Kumar, Proc. R. Soc. London Ser. B 169, 167 by using transgenic medaka harboring two dis- Movies S1 to S3 hsp (1968). tinct fluorescent constructs { -cre:mCherry/ 3. J. Johnson, J. Canning, T. Kaneko, J. K. Pru, J. L. Tilly, 3 December 2009; accepted 5 May 2010 nos2p-loxP[DsRed]-EGFP-olvas3′ untranslated Nature 428, 145 (2004). Published online 20 May 2010; region (UTR)} (fig. S8). In these medaka, heat 4. K. Zou et al., Nat. Cell Biol. 11, 631 (2009). 10.1126/science.1185473 treatment transiently induces EGFP expression 5. J. Pacchiarotti et al., Differentiation 79, 159 (2010). Include this information when citing this paper. only in nos2-expressing Gs cells, and EGFP tran- scripts are stabilized in all germ cells through the ′ olvas 14 15 action of the 3 UTR of ( , ). Downloaded from Adult transgenic fish (n = 21) were heated to Down-Regulation of a Host 39°C for 2 hours, and immunohistochemistry was performed by using antibodies specific for MicroRNA by a Herpesvirus saimiri GFP and the pan–germ cell marker tdrd1 (16). Several different combinations of EGFP fluores- Noncoding RNA cent cells were found in the germinal cradles, in- cluding cradles in which the only EGFP-labeled Demián Cazalla, Therese Yario, Joan Steitz* germ cells were Gs cells (Gs clone type) (Fig. 2A and fig. S9, E and F), cradles containing fluores- T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function cent Gs cells as well as Gcys cells and/or Gdip called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites oocytes (mosaic clone type) (Fig. 2B and fig. S9, for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 G and H), and cradles entirely occupied by flu- and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these orescent germ cells (full clone type) (Fig. 2C and miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of fig. S9, I and J). Forty-eight hours after heat treat- miR-27 target genes.
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