Maternal factor-mediated epigenetic gene silencing in the ascidian Ciona intestinalis

  • Yasunori SasakuraEmail author
  • Miho M. Suzuki
  • Akiko Hozumi
  • Kazuo Inaba
  • Nori Satoh
Original Paper


Epigenetic regulation of genes plays a critical role in achieving proper gene expression during development, and it has been reported that epigenetic modifications are associated with transposon silencing in many organisms. Here, we report a type of epigenetic gene silencing, maternal gfp/gene silencing (MGS), in the basal chordate Ciona intestinalis. A transgenic line of Ciona, Tg[MiTFr3dTPOG]45 (abbreviated as Tg45), which was created with the Minos transposon, has a tandemly arrayed insertion of gfp in the promoter region of Ci-CesA. Progeny of Tg45 showed a reduced level of GFP expression when eggs of Tg45 were fertilized with sperm of other gfp transgenic lines. Although the genotype is the same, animals developed from Tg45 sperm and the eggs of other transgenic lines did not exhibit this phenomenon, suggesting the involvement of a maternal cytoplasmic factor that influences GFP expression. The silencing starts during oogenesis and continues after fertilization without any tissue specificity. We found that post-transcriptional degradation of the gfp mRNA is involved in MGS.


Epigenetics Minos Ci-CesA Transcription 



The authors would like to thank Kazuko Hirayama, Yasuo Kasuga, Yasutaka Tsuchiya, Toshihiko Sato, Hideo Shinagawa, Yoshiko Harada and Hiromi Takahashi for their kind cooperation in our study. We thank all members of the Maizuru Fishery Research Station of Kyoto University, the International Coast Research Center of the Ocean Research Institute of the University of Tokyo and the Education and Research Center of Marine Bioresources of Tohoku University for the collection of Ciona adults. We also thank Shigeki Fujiwara and his laboratory members for useful discussion and for providing wild-type Ciona. We are grateful to William Smith, Charalambos Savakis, Brad Davidson, Maki Shirae-Kurabayashi, Hiroki Takahashi, Takahito Nishikata, Naohito Takatori and Hiroki Nishida for kindly providing Cs-CesA cDNA, Minos, pMiCiTnIG, pMiCiEf1aG, pCi-Bra promoter/pEGFP-N1/CMV(−), pCi-Nut promoter/pEGFP-N1/CMV(−) and pBS-HTB. We are also grateful to Adrian Bird for his helpful discussions and for generously allowing the use of his faculty. This study was supported by Grants-in-Aid for Scientific Research from JSPS and MEXT to Nori Satoh (17207013) and Yasunori Sasakura (18770193 and 20681019). Yasunori Sasakura was also supported by the NIG Cooperative Research Program (2007-B01, 2008-B02 and 2009-A18) and by a Narishige Zoological Science Award. This study was further supported by grants from the National Bioresource Project.

Supplementary material

438_2009_500_MOESM1_ESM.pdf (2.8 mb)
Supplementary material 1 (PDF 2904 kb)


  1. Akanuma T, Hori S, Darras S, Nishida H (2002) Notch signaling is involved in neurogenesis in the ascidian embryos. Dev Genes Evol 212:459–472CrossRefPubMedGoogle Scholar
  2. Awazu S, Sasaki A, Matsuoka T, Satoh N, Sasakura Y (2004) An enhancer trap in the ascidian Ciona intestinalis identifies enhancers of its Musashi orthologous gene. Dev Biol 275:459–472CrossRefPubMedGoogle Scholar
  3. Awazu S, Matsuoka T, Inaba K, Satoh N, Sasakura Y (2007) High-throughput enhancer trap by remobilization of transposon Minos in Ciona intestinalis. Genesis 45:307–317CrossRefPubMedGoogle Scholar
  4. Bird AP, Wolffe AP (1999) Methylation-induced repression—belts, braces, and chromatin. Cell 99:451–454CrossRefPubMedGoogle Scholar
  5. Blumenstiel JP, Hartl DL (2005) Evidence for maternally transmitted small interfering RNA in the repression of transposition in Drosophila virilis. Proc Natl Acad Sci USA 102:15965–15970CrossRefPubMedGoogle Scholar
  6. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 162:156–159CrossRefPubMedGoogle Scholar
  7. Corbo JC, Levine M, Zeller RW (1997) Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124:589–602PubMedGoogle Scholar
  8. Dehal P, Satou Y et al (2002) The draft genome of Ciona intestinalis: insight into chordate and vertebrate origins. Science 298:2157–2167CrossRefPubMedGoogle Scholar
  9. Franz G, Savakis CC (1991) Minos, a new transposable element from Drosophila hydei, is a member of the Tc1-like family of transposons. Nucleic Acid Res 19:6646CrossRefPubMedGoogle Scholar
  10. Girard A, Hannon GJ (2008) Conserved themes in small-RNA-mediated transposon control. Trends Cell Biol 18:136–148CrossRefPubMedGoogle Scholar
  11. Holdeman R, Nehrt S, Strome S (1998) MES-2, a maternal protein essential for viability of the germline in C. elegans, is homologous to a Drosophila polycomb group protein. Development 125:2457–2467PubMedGoogle Scholar
  12. Hotta K, Yamada S, Ueno N, Satoh N, Takahashi H (2007) Brachyury-downstream notochord genes and convergent extension in Ciona intestinalis embryos. Dev Growth Differ 49:373–382PubMedGoogle Scholar
  13. Joly JS, Kano S, Matsuoka T, Auger H, Hirayama K, Satoh N, Awazu S, Legendre L, Sasakura Y (2007) Culture of Ciona intestinalis in closed systems. Dev Dyn 236:1832–1840CrossRefPubMedGoogle Scholar
  14. Kelly WG, Fire A (1998) Chromatin silencing and the maintenance of a functional germline in Caenorhabditis elegans. Development 125:2451–2456PubMedGoogle Scholar
  15. Kitaura Y, Sonobe H, Tanaka O, Honda D, Watanabe Y, Makabe KW, Takamura K, Nishikata T (2007) Neural-tube specific paralogous genes and their upstream regulatory sequences. Mem Konan Univ Sci Eng Ser 54:75–88Google Scholar
  16. Klinakis AG, Loukeris TG, Pavlopoulos A, Savakis C (2000) Mobility assays confirm the broad host-range activity of the Minos transposable element and validate new transformation tools. Insect Mol Biol 9:269–275CrossRefPubMedGoogle Scholar
  17. Kusakabe T, Yoshida R, Ikeda Y, Tsuda M (2004) Computational discovery of DNA motifs associated with cell type-specific gene expression in Ciona. Dev Biol 276:563–580CrossRefPubMedGoogle Scholar
  18. Martienssen RA (2003) Maintenance of heterochromatin by RNA interference of tandem repeats. Nat Genet 35:213–214CrossRefPubMedGoogle Scholar
  19. Matsuoka T, Awazu S, Shoguchi E, Satoh N, Sasakura Y (2005) Germline transgenesis of the ascidian Ciona intestinalis by electroporation. Genesis 41:61–72CrossRefGoogle Scholar
  20. Matthysse AG, Deschet K, Williams M, Marry M, White AR, Smith WC (2004) A functional cellulose synthase from ascidian epidermis. Proc Natl Acad Sci USA 101:986–991CrossRefPubMedGoogle Scholar
  21. Matzke M, Aufsatz W, Kanno T, Daxinger L, Papp I, Mette MF, Matzke AJM (2004) Genetic analysis of RNA-mediated transcriptional gene silencing. Biochim Biophys Acta 1677:129–141PubMedGoogle Scholar
  22. Miya T, Makabe KW, Satoh N (1994) Expression of a gene for major mitochondrial protein, ADP/ATP translocase, during embryogenesis in the ascidian Halocynthia roretzi. Dev Growth Differ 36:39–48CrossRefGoogle Scholar
  23. Mochizuki K, Gorovsky MA (2004) Small RNAs in genome rearrangement in Tetrahymena. Curr Opin Genet Dev 14:181–187CrossRefPubMedGoogle Scholar
  24. Nakashima K, Yamada L, Satou Y, Azuma J, Satoh N (2004) The evolutionary origin of animal cellulose synthase. Dev Genes Evol 214:81–88CrossRefPubMedGoogle Scholar
  25. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279–289CrossRefPubMedGoogle Scholar
  26. Sasakura Y (2007) Germline transgenesis and insertional mutagenesis in the ascidian Ciona intestinalis. Dev Dyn 236:1758–1767CrossRefPubMedGoogle Scholar
  27. Sasakura Y, Awazu S, Chiba S, Kano S, Satoh N (2003a) Application of Minos, one of the Tc1/mariner superfamily transposable elements, to ascidian embryos as a tool for insertional mutagenesis. Gene 308:11–20CrossRefPubMedGoogle Scholar
  28. Sasakura Y, Awazu S, Chiba S, Satoh N (2003b) Germ-line transgenesis of the Tc1/mariner superfamily transposon Minos in Ciona intestinalis. Proc Natl Acad Sci USA 100:7726–7730CrossRefPubMedGoogle Scholar
  29. Sasakura Y, Nakashima K, Awazu S, Matsuoka T, Nakayama A, Azuma J, Satoh N (2005) Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc Natl Acad Sci USA 102:15134–15139CrossRefPubMedGoogle Scholar
  30. Sasakura Y, Oogai Y, Matsuoka T, Satoh N, Awazu S (2007) Transposon-mediated transgenesis in a marine invertebrate chordate, Ciona intestinalis. Genome Biol 8:S3CrossRefPubMedGoogle Scholar
  31. Sasakura Y, Konno A, Mizuno K, Satoh N, Inaba K (2008) Enhancer detection in the ascidian Ciona intestinalis with transposase-expressing lines of Minos. Dev Dyn 237:39–50CrossRefPubMedGoogle Scholar
  32. Suzuki MM, Kerr AR, De Sousa D, Bird A (2007) CpG methylation is targeted to transcription units in an invertebrate genome. Genome Res 17:625–631CrossRefPubMedGoogle Scholar
  33. Wakimoto BT (1998) Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila. Cell 93:321–324CrossRefPubMedGoogle Scholar
  34. Yamada L, Saito T, Taniguchi H, Sawada H, Harada Y (2009) Comprehensive egg-coat proteome of an ascidian Ciona intestinalis reveals gamete recognition molecules involved in self-sterility. J Biol Chem 284:9402–9410CrossRefPubMedGoogle Scholar
  35. Yasuo H, Satoh N (1994) An ascidian homolog of the mouse Brachyury (T) gene is expressed exclusively in notochord cells at the fate restricted stage. Dev Growth Differ 36:9–18CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Yasunori Sasakura
    • 1
    Email author
  • Miho M. Suzuki
    • 2
  • Akiko Hozumi
    • 1
  • Kazuo Inaba
    • 1
  • Nori Satoh
    • 3
  1. 1.Shimoda Marine Research CenterUniversity of TsukubaShimodaJapan
  2. 2.The Wellcome Trust Centre for Cell BiologyThe University of EdinburghEdinburghUK
  3. 3.Marine Genomics UnitOkinawa Institute of Science and Technology Promotion CorporationUrumaJapan

Personalised recommendations