Development Genes and Evolution

, Volume 216, Issue 2, pp 69–80 | Cite as

The functional analysis of Type I postplasmic/PEM mRNAs in embryos of the ascidian Halocynthia roretzi

  • Yoriko NakamuraEmail author
  • Kazuhiro W. Makabe
  • Hiroki Nishida
Original Article


Maternal factors, such as a muscle determinant macho-1 mRNA that is localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, are crucial for embryonic axis formation and cell fate specification. Maternal mRNAs that show an identical posterior localization pattern to that of macho-1 in eggs and embryos are called Type I postplasmic/PEM mRNAs. We investigated the functions of five of the nine Type I mRNAs so far known in Halocynthia roretzi: Hr-Wnt-5, Hr-GLUT, Hr-PEM3, Hr-PEN1, and Hr-PEN2. Suppression of their functions with specific antisense morpholino oligonucleotides (MOs) had effects on the formation of various tissues: Hr-Wnt-5 on notochord, muscle, and mesenchyme, although zygotic function of Hr-Wnt-5 is responsible for notochord formation; Hr-GLUT on notochord, mesenchyme, and endoderm; and Hr-PEN2 on muscle, mesenchyme, and endoderm. On the other hand, Hr-PEM3 and Hr-PEN1 MOs seemed to have no effect. We conclude that the functions of at least some localized maternal Type I postplasmic/PEM mRNAs are necessary for early embryonic patterning in ascidians.


Ascidian embryo Maternal RNA localization Type I postplasmic/PEM RNA Wnt Embryonic patterning 



We thank the staff of the Asamushi Research Center for Marine Biology and the Otsuchi International Coastal Research Center for help in collecting live ascidian adults, and the staff of the Misaki Marine Biological Station and Seto Marine Biological Laboratory for help in maintaining them. We also thank Dr. Nori Satoh for providing HrBra cDNA, Dr. T. Nishikata for the Not-1, Mu-2, and Epi-2 monoclonal antibodies, Dr. Y. Sasakura for Hr-Wnt-5 cDNA, and Drs. T. Kawashima and Y. Kohara for MAGEST plasmids. This work was supported by Grants in Aid from MEXT (13044003) and JSPS (13480245 and 16107005) and by Toray Science and Technology Grant.


  1. Akanuma T, Hori S, Darras S, Nishida H (2002) Notch signaling is involved in nervous system formation in ascidian embryos. Dev Genes Evol 212:459–472CrossRefPubMedGoogle Scholar
  2. Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer EL, Studholme DJ, Yeats C, Eddy SR (2004) The Pfam protein families database. Nucleic Acids Res 32:138–141CrossRefGoogle Scholar
  3. Christian JL, Moon RT (1993) Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes Dev 7:13–28PubMedCrossRefGoogle Scholar
  4. Darras S, Nishida H (2001) The BMP signaling pathway is required together with the FGF pathway for notochord induction in the ascidian embryo. Development 128:2629–2638PubMedGoogle Scholar
  5. Heasman J, Kofron M, Wylie C (2000) Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev Biol 222:124–134CrossRefPubMedGoogle Scholar
  6. Hibino T, Nishikata T, Nishida H (1998) Centrosome-attracting body: a novel structure closely related to unequal cleavages in the ascidian embryo. Dev Growth Differ 40:85–95CrossRefPubMedGoogle Scholar
  7. Hino K, Satou Y, Yagi K, Satoh N (2003) A genomewide survey of developmentally relevant genes in Ciona intestinalis. VI. Genes for Wnt, TGFβ, Hedgehog and JAK/STAT signaling pathways. Dev Genes Evol 213:264–272CrossRefPubMedGoogle Scholar
  8. Hopper S, Moon RT (1998) BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm. Mech Dev 71:119–129CrossRefPubMedGoogle Scholar
  9. Huelsken J, Birchmeier W (2001) New aspects of Wnt signaling pathways in higher vertebrates. Curr Opin Genet Dev 11:547–553CrossRefPubMedGoogle Scholar
  10. Hulo N, Sigrist CJ, Le Saux V, Langendijk-Genevaux PS, Bordoli L, Gattiker A, De Castro E, Bucher P, Bairoch A (2004) Recent improvements to the PROSITE database. Nucleic Acids Res 32:134–137CrossRefGoogle Scholar
  11. Imai KS, Satoh N, Satou Y (2002) Early embryonic expression of FGF4/6/9 gene and its role in the induction of mesenchyme and notochord in Ciona savignyi embryos. Development 129:1729–1738PubMedGoogle Scholar
  12. Iseto T, Nishida H (1999) Ultrastructural studies on the centrosome-attracting body: electron-dense matrix and its role in unequal cleavages in ascidian embryos. Dev Growth Differ 41:601–609CrossRefPubMedGoogle Scholar
  13. Karnovsky MJ, Roots L (1964) A ‘direct-coloring’ thiocholine method for cholinesterase. J Histochem Cytochem 12:219–221PubMedGoogle Scholar
  14. Kawashima T, Kawashima S, Kanehisa M, Nishida H, Makabe KW (2000) MAGEST: Maboya gene expression patterns and sequence tags. Nucleic Acids Res 28:133–135CrossRefPubMedGoogle Scholar
  15. Kim GJ, Nishida H (1998) Monoclonal antibodies against differentiating mesenchyme cells in larvae of the ascidian, Halocynthia roretzi. Zool Sci 15:553–559CrossRefPubMedGoogle Scholar
  16. Kim GJ, Nishida H (1999) Suppression of muscle fate by cellular interaction is required for mesenchyme formation during ascidian embryogenesis. Dev Biol 214:9–22CrossRefPubMedGoogle Scholar
  17. Kim GJ, Yamada A, Nishida H (2000) An FGF signal from endoderm and localized factors in the posterior-vegetal egg cytoplasm pattern the mesodermal tissues in the ascidian embryo. Development 127:2853–2862PubMedGoogle Scholar
  18. Kobayashi K, Sawada K, Yamamoto H, Wada S, Saiga H, Nishida H (2003) Maternal macho-1 is an intrinsic factor that makes cell response to the same FGF signal differ between mesenchyme and notochord induction in ascidian embryos. Development 130:5179–5190CrossRefPubMedGoogle Scholar
  19. Kondoh K, Kobayashi K, Nishida H (2003) Suppression of macho-1-directed muscle fate by FGF and BMP is required for formation of posterior endoderm in ascidian embryos. Development 130:3205–3216CrossRefPubMedGoogle Scholar
  20. Letunic I, Copley RR, Schmidt S, Ciccarelli FD, Doerks T, Schultz J, Ponting CP, Bork P (2004) SMART 4.0: towards genomic data integration. Nucleic Acids Res 32:142–144CrossRefGoogle Scholar
  21. Makabe KW, Satoh N, (1989) Temporal expression of myosin heavy chain gene during ascidian embryogenesis. Dev Growth Differ 31:71–77CrossRefGoogle Scholar
  22. Makabe KW, Kawashima T, Kawashima S, Minokawa T, Adachi A, Kawamura H, Ishikawa H, Yasuda R, Yamamoto H, Kondoh K, Arioka S, Sasakura Y, Kobayashi A, Yagi K, Shojima K, Kondoh Y, Kido S, Tsujinami M, Nishimura N, Takahashi M, Nakamura T, Kanehisa M, Ogasawara M, Nishikata T, Nishida H (2001) Large-scale cDNA analysis of the maternal genetic information in the egg of Halocynthia roretzi for a gene expression catalog of ascidian development. Development 128:2555–2567PubMedGoogle Scholar
  23. Marchler-Bauer A, Anderson JB, Cherukuri PF, DeWeese-Scott C, Geer LY, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Liebert CA, Liu C, Lu F, Marchler GH, Mullokandov M, Shoemaker BA, Simonyan V, Song JS, Thiessen PA, Yamashita RA, Yin JJ, Zhang D, Bryant SH (2005) CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res 33(Database issue):D192–D196CrossRefPubMedGoogle Scholar
  24. Minokawa T, Yagi K, Makabe KW, Nishida H (2001) Binary specification of nerve cord and notochord cell fates in ascidian embryos. Development 128:2007–2017PubMedGoogle Scholar
  25. Miya T, Nishida H (2002) Isolation of cDNA clones for mRNAs transcribed zygotically during cleavage in the ascidian, Halocynthia roretzi. Dev Genes Evol 212:30–37CrossRefPubMedGoogle Scholar
  26. 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
  27. Miya T, Morita K, Suzuki A, Ueno N, Satoh N (1997) Functional analysis of an ascidian homologue of vertebrate Bmp-2/Bmp-4 suggests its role in the inhibition of neural fate specification. Development 124:5149–5159PubMedGoogle Scholar
  28. Nakamura Y, Makabe KW, Nishida H (2003) Localization and expression pattern of type I postplasmic mRNAs in embryos of the ascidian Halocynthia roretzi. Gene Expr Patterns 3:71–75CrossRefPubMedGoogle Scholar
  29. Nakatani Y, Nishida H (1994) Induction of notochord during ascidian embryogenesis. Dev Biol 166:289–299CrossRefPubMedGoogle Scholar
  30. Nakatani Y, Yasuo H, Satoh N, Nishida H (1996) Basic fibroblast growth factor induces notochord formation and the expression of As-T, a Brachyury homolog, during ascidian embryogenesis. Development 122:2023–2031PubMedGoogle Scholar
  31. Nishida H (1987) Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev Biol 121:526–541CrossRefPubMedGoogle Scholar
  32. Nishida H (1994) Localization of determinants for formation of the anterior–posterior axis in eggs of the ascidian Halocynthia roretzi. Development 120:3093–3104Google Scholar
  33. Nishida H (1997) Cell fate specification by localized cytoplasmic determinants and cell interactions in ascidian embryos. Int Rev Cytol 176:245–306PubMedCrossRefGoogle Scholar
  34. Nishida H (2002) Specification of developmental fates in ascidian embryos: molecular approach to maternal determinants and signaling molecules. Int Rev Cytol 217:227–276PubMedGoogle Scholar
  35. Nishida H, Sawada K (2001) macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis. Nature 409:724–729CrossRefPubMedGoogle Scholar
  36. Nishikata T, Satoh N (1990) Specification of notochord cells in the ascidian embryo analysed with a specific monoclonal antibody. Cell Differ Dev 30:43–53CrossRefPubMedGoogle Scholar
  37. Nishikata T, Mita-Miyazawa I, Deno T, Satoh N (1987a) Muscle cell differentiation in ascidian embryos analysed with a tissue-specific monoclonal antibody. Development 99:163–171PubMedGoogle Scholar
  38. Nishikata T, Mita-Miyazawa I, Deno T, Takamura K, Satoh N (1987b) Expression of epidermis-specific antigens during embryogenesis of the ascidian, Halocynthia roretzi. Dev Biol 121:408–416CrossRefPubMedGoogle Scholar
  39. Nishikata T, Hibino T, Nishida H (1999) The centrosome-attracting body, microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo. Dev Biol 209:72–85CrossRefPubMedGoogle Scholar
  40. Pandur P, Maurus D, Kuhl M (2002) Increasingly complex: new players enter the Wnt signaling network. Bioessays 24:881–884CrossRefPubMedGoogle Scholar
  41. Sardet C, Nishida H, Prodon F, Sawada K (2003) Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex. Development 130:5839–5849CrossRefPubMedGoogle Scholar
  42. Sardet C, Dru P, Prodon F (2005) Maternal determinants and mRNA in the cortex of ascidian oocytes, zygotes and embryos. Biol Cell 97:1–15CrossRefPubMedGoogle Scholar
  43. Sasakura Y, Makabe KW (2001) Ascidian Wnt-5 gene is involved in the morphogenetic movement of notochord cells. Dev Growth Differ 43:573–582CrossRefPubMedGoogle Scholar
  44. Sasakura Y, Ogasawara M, Makabe KW (1998a) Hr-Wnt-5: a maternally expressed ascidian Wnt gene with posterior localization in early embryos. Int J Dev Biol 42:573–579PubMedGoogle Scholar
  45. Sasakura Y, Ogasawara M, Makabe KW (1998b) Maternally localized RNA encoding a serine/threonine protein kinase in the ascidian, Halocynthia roretzi. Mech Dev 76:161–163CrossRefPubMedGoogle Scholar
  46. Sasakura Y, Ogasawara M, Makabe KW (2000) Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos. Dev Biol 220:365–378CrossRefPubMedGoogle Scholar
  47. Satou Y (1999) posterior end mark 3 (pem-3), an ascidian maternally expressed gene with localized mRNA encodes a protein with Caenorhabditis elegans MEX-3-like KH domains. Dev Biol 212:337–350CrossRefPubMedGoogle Scholar
  48. Satou Y, Imai KS, Satoh N (2001a) Action of morpholinos in Ciona embryos. Genesis 30:103–106CrossRefPubMedGoogle Scholar
  49. Satou Y, Imai KS, Satoh N (2001b) Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream of beta-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos. Development 128:3559–3570PubMedGoogle Scholar
  50. Sawada K, Fukushima Y, Nishida H (2005) Macho-1 functions as transcriptional activator for muscle formation in embryos of the ascidian Halocynthia roretzi. Gene Expr Patterns 5:429–437CrossRefPubMedGoogle Scholar
  51. Sheldahl LC, Park M, Malbon CC, Moon RT (1999) Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr Biol 9:695–698CrossRefPubMedGoogle Scholar
  52. Shimauchi Y, Murakami SD, Satoh N (2001) FGF signals are involved in the differentiation of notochord cells and mesenchyme cells of the ascidian Halocynthia roretzi. Development 128:2711–2721PubMedGoogle Scholar
  53. Slusarski DC, Yang-Snyder J, Busa WB, Moon RT (1997) Modulation of embryonic intracellular Ca2+ signaling by Wnt-5A. Dev Biol 182:114–120CrossRefPubMedGoogle Scholar
  54. Takamura K, Fujimura M, Yamaguchi Y (2002) Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis. Dev Genes Evol 212:11–18CrossRefPubMedGoogle Scholar
  55. Whittaker JR (1973) Segregation during ascidian embryogenesis of egg cytoplasmic information for tissue-specific enzyme development. Proc Natl Acad Sci U S A 70:2096–2100PubMedCrossRefGoogle Scholar
  56. Whittaker JR, Meedel TH (1989) Two histospecific enzyme expression in the same cleavage-arrested one-celled ascidian embryos. J Exp Zoolog 250:168–175CrossRefGoogle Scholar
  57. Wodarz A, Nusse R (1998) Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 14:59–88CrossRefPubMedGoogle Scholar
  58. Yasuo H, Satoh N (1993) Function of vertebrate T gene. Nature 364:582–583CrossRefPubMedGoogle Scholar
  59. Yasuo H, Lemaire P (2001) Role of Goosecoid, Xnot and Wnt antagonists in the maintenance of the notochord genetic programme in Xenopus gastrulae. Development 128:3783–3793PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Yoriko Nakamura
    • 1
    Email author
  • Kazuhiro W. Makabe
    • 2
  • Hiroki Nishida
    • 1
  1. 1.Department of Biological SciencesGraduate School of Science, Osaka UniversityOsakaJapan
  2. 2.Faculty of Integrated Arts and SciencesTokushima UniversityTokushimaJapan

Personalised recommendations